Cancer test device, cancer test method, and staining agent for use in cancer test

ABSTRACT

A cancer test device (1) is provided with: an application unit (40) for applying a staining agent (45), which can selectively stain a product of a cancer-relating gene in a living cell a chromatic color, onto a group of living cells; an imaging unit (10) for imaging the group of living cells having the staining agent (45) applied thereto; and a determination unit (52) for determining the level of malignancy of cancerization of the group of living cells on the basis of the state of the stained expression pattern of the cancer-relating gene in the group of living cells in an image obtained by the aforementioned imaging.

TECHNICAL FIELD

The present invention relates to a cancer test device and a cancer testmethod for testing a cancerous living cell and a stain for use in acancer test.

BACKGROUND ART

In recent years, as a method for checking a lesion in a living body(digestive tract, for example), there is a known method for imaging theform of a cell group in the living body to check whether or not alesion, such as cancer cells, is present.

As an example of the method, Patent Literature 1 describes a method forstaining a predetermined cell group in the living body in living bodystaining using a specific edible dye, such as curcumin and sulfuretin,and then applying multiphoton laser light to the stained cell group toreadily detect the cancer cells because cancer cells are stained moreheavily than healthy cells and further capture a fluorescence image ofthe individual cell forms in the living body. According to the methoddescribed above, since the cell group stained the living body emitsfluorescence when the multiphoton laser light is applied thereto, asharp image of the forms of the individual cells and nuclei in theliving body can be generated. Whether or not a lesion, such as cancercells, is present can therefore be precisely checked for pathologicaldiagnosis.

CITATION LIST Patent Literature

Patent Literature 1: International Publication No. WO 2014/157703

SUMMARY OF INVENTION Technical Problem

The method described in Patent Literature 1 can be used to preciselycheck whether or not cancerization of a living cell has occurred. It is,however, required as a public requirement to grasp cancerization of aliving cell as soon as possible.

The present invention achieves the requirement described above, and anobject of the present invention is to provide a cancer test device andthe like capable of grasping cancerization of a living cell in an earlystage.

Solution to Problem

To achieve the object described above, a cancer test device according toan aspect of the present invention includes (1) an application unit thatapplies a stain to a living cell group, the stain selectively staining acancer-related gene product of living cells in a chromatic color, (2) animaging unit that images the living cell group to which the stain hasbeen applied, and (3) an evaluation unit that evaluates, by theexpression pattern, a grade of cancerization of the living cell groupbased on a staining state of the living cell group in an image producedby the imaging. A laser used in the imaging may be a multiphoton laserfor a multiphoton laser microscope or a continuous-wave (CW) laser for aconfocal laser microscope. The grade of cancerization used herein is sodefined that a cancer cell having a high degree of the metastasis andinfiltration capability, which the cancer cell inherently possesses, hasa high grade of cancerization and cancer resistant to radiotherapy andchemotherapy has a high grade of cancerization.

According to the present aspect, the cancer test device evaluates thegrade of cancerization based on the staining state of the cancer-relatedgene product of the living cell group, whereby cancerization of theliving cell group can be grasped in an early stage. Further, since thegrade of cancerization can be grasped, the prognosis of the cancerpatient can be understood.

For example, the application unit may apply the stain that stains aras-family cancer-related gene product that transmits a signal thatpromotes growth of the living cells.

Using the stain that stains the ras-family cancer-related gene productallows understanding of the tendency of the growth of the living cells,as in the present aspect, whereby development of cancer cells in theliving cell group can be grasped in an early stage.

For example, the application unit may apply the stain containingphloxine, erythrosine, merbromin, fast green FCF, or meclocyclinesulfosalicylate.

Using the stain shown in the present aspect allows staining of theras-family cancer-related gene product, whereby development of cancer inthe living cell group can be grasped in an early stage.

For example, the application unit may apply the stain that stains aSTAT3-family cancer-related gene product that transmits a signal thatpromotes growth of the living cells.

Using the stain that stains the STAT3-family cancer-related gene productallows understanding of the tendency of the growth of the living cells,as in the present aspect, whereby development of cancer cells in theliving cell group can be grasped in an early stage.

For example, the application unit may apply the stain containing acurcumin-based compound.

Using the stain shown in the present aspect allows staining of theSTAT3-family cancer-related gene product, whereby development of cancercells in the living cell group can be grasped in an early stage.

For example, the application unit may apply the stain containing acurcumin-based compound to the living cell group and then apply thestain containing phloxine, erythrosine, merbromin, fast green FCF, ormeclocycline sulfosalicylate to the living cell group.

Applying the stain containing a curcumin-based compound before applyingthe stain containing phloxine, erythrosine, merbromin, fast green FCF,or meclocycline sulfosalicylate allows the contour of each of the cellsand the shape of the nucleus in the cell to be clearly visualized,whereby a sharp image can be produced.

For example, the application unit may apply to the living cell group thestain that stains a STAT3-family cancer-related gene product thattransmits a signal that promotes growth of the living cells and thenapply the stain that stains a ras-family cancer-related gene productthat transmits a signal that promotes growth of the living cells.

Applying the stain that stains the STAT3-family cancer-related geneproduct before applying the stain that stains the ras-familycancer-related gene product allows the STAT3-family cancer-related geneproduct to be clearly visualized, whereby a sharp image can be produced.

For example, the evaluation unit may perform the evaluation based on anarea of a stained region of the living cell group.

According to the present aspect, the state of enhancement of expressionof the cancer-related gene can be understood based on the area of thestained region, whereby the grade of cancerization can be preciselygrasped.

For example, the evaluation unit may perform the evaluation based on thenumber of cells in a stained region of the living cell group.

According to the present aspect, the state of enhancement of expressionof the cancer-related gene can be understood based on the number ofcells in the stained region, whereby the grade of cancerization can beprecisely grasped.

For example, the evaluation unit may perform the evaluation based on thenumber and average diameter of stained cell groups in a fixed areacontaining a stained region of the living cell group.

According to the present aspect, the state of enhancement of expressionof the cancer-related gene can be understood based on the number and theaverage diameter of stained cell groups in the fixed area, whereby thegrade of cancerization can be precisely grasped.

For example, the imaging unit may irradiate the living cell group towhich the stain has been applied with multiphoton laser light orconfocal laser light and image the living cell group.

Irradiating the living cell group with multiphoton laser light, as inthe present aspect, allows the grade of cancerization to be readilygrasped over the depth range greater than or equal to 10 μm but smallerthan or equal to 1000 μm below the mucosa surface in the living body.Further, irradiating the living cell group with confocal laser lightallows the grade of cancerization to be readily grasped over the depthrange greater than or equal to 10 μm but smaller than or equal to 70 μmbelow the mucosa surface in the living body. The prognosis of the cancerpatient can therefore be understood in an ultra-early stage before acancer cell population appears on the mucosa surface.

For example, the imaging unit may image the cancer-related geneexpression pattern of the cell population stained with the stain andhaving a diameter greater than or equal to 0.1 mm but smaller than orequal to 0.4 mm.

According to the present aspect, the grade of cancerization of theliving cells in a pre-cancer state can be grasped, whereby the prognosisof the cancer patient can be understood in an early stage before acancer cell population clearly manifests.

For example, the application unit may apply a plurality of differentstains to the living cell group to stain a plurality of cancer-relatedgene products in colors different from one another, and the imaging unitmay irradiate the plurality of cancer-related gene expression patternsstained in the different colors with a plurality of excitation lightbeams according to the stains and image the plurality of cancer-relatedgene expression patterns.

Irradiating the plurality of stained cancer-related gene expressionpatterns with the plurality of excitation light beams according to thestains allows precise detection of the plurality of cancer-related geneexpression patterns.

For example, the stains may be formed of at least two types of stain,and the excitation light beams with which the plurality ofcancer-related gene expression patterns are irradiated may be selectedin correspondence with the types of the stain.

Using at least two types of stain and irradiating the plurality ofcancer-related gene expression patterns with excitation light beamscorresponding to the stains allows detection of at least twocancer-related gene expression patterns. Detecting the manycancer-related gene expression patterns as described above allows thegrade of cancerization to be grasped from diverse viewpoints.

For example, the imaging unit may include a focal point position controlunit and control the focal point position control unit to image thecancer-related gene expression pattern present over the depth rangegreater than or equal to 10 μm but smaller than or equal to 1000 below asurface in the living body stained with the stain.

According to the present aspect, the grade of cancerization of theinterior of the living body over the depth range greater than or equalto 10 μm but smaller than or equal to 1000 μm below the mucosa surfacecan be grasped, whereby the prognosis of the cancer patient can beunderstood in an early stage before a cancer cell population appears onthe mucosa surface.

For example, the focal point position control unit may be controlled tochange a focal point at fixed intervals from the surface in the sameimaging position in the living body stained with the stain to performimaging at different-depth focal point positions, a plurality ofcaptured images may be superimposed on each other in a focal pointposition information order into a stereoscopic image, and the evaluationmay be performed based on a degree of penetration of the stain in thestereoscopic image.

According to the present aspect, the grade of cancerization of theinterior of the living body can be grasped based on the degree ofpenetration of the stain in the stereoscopic image, whereby theprognosis of the cancer patient can be understood in an ultra-earlystage before a cancer cell population appears on the mucosa surface.

A cancer test method according to another aspect of the presentinvention includes an application step of applying a stain to a livingcell group, the stain selectively staining a cancer-related gene productof living cells in a chromatic color, an imaging step of imaging theliving cell group to which the stain has been applied, and an evaluationstep of evaluating a grade of cancerization of the living cell groupbased on a staining state of the living cell group in an image producedby the imaging.

According to the present aspect, the cancer test method evaluates thegrade of cancerization based on the staining state of the cancer-relatedgene expression pattern of the living cell group, whereby cancerizationof the living cell group can be grasped in an early stage. Further,since the grade of cancerization can be grasped, the prognosis of thecancer patient can be understood.

A stain for a cancer test according to another aspect of the presentinvention contains phloxine, erythrosine, merbromin, fast green FCF, ormeclocycline sulfosalicylate, which stains a ras-family cancer-relatedgene product that transmits a signal that promotes growth of livingcells, or a curcumin-based compound, which stains a STAT3-familycancer-related gene product that transmits a signal that promotes growthof the living cells, and the stain has a concentration that allows thestain to penetrate into a cytoplasm of the living cells within 10minutes after the staining starts but does not penetrate into a nucleusof the cells.

Since the stain for a cancer test according to the present aspectpenetrates into the cytoplasm but does not penetrate into the nucleus ofthe cells as long as the period having elapsed since the stainingstarted is 10 minutes or shorter, the nucleus surrounded by thecytoplasm can be sharply visualized, whereby analysis of cancerizationcan be more distinctly performed.

Advantageous Effects of Invention

According to the present invention, cancerization of living cell can begrasped in a very early stage at which a cancerous portion has adiameter of about 1 mm or in an ultra-early stage.

Further, according to the primary configuration of the presentinvention, the expression pattern of a cancer-related gene can beanalyzed, whereby the degree of risk that a cancer tumor affects thepatient (vital prognosis) can be determined.

The observation target described above only includes the epithelialcells, the glandular cells, connective tissue, and capillaries in themucosa surface on the inner wall of a digestive tract of a living body.Instead, fresh tissue within 20 minutes immediately after the tissue issurgically excised can be used as the observation material to image acell form image similar to an image of living tissue.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a diagrammatic view showing a state in which cancer-relatedgene products in a living cell act normally.

FIG. 1B is a diagrammatic view showing a state in which thecancer-related gene products in the living cell act abnormally. Thecross star marks in FIG. 1B each indicate cancerous mutation that occursin the corresponding cancer-related gene product.

FIG. 1C is a diagrammatic view showing a stepwise cancerization processof a living cell group on the inner wall surface of a digestive tract.

FIG. 1D shows an example of a human cancer cell growth curve.

FIG. 2A shows images of a cancer-related gene expression pattern ofliving cells associated with a specimen 1: The section (a) shows animage of an STAT3 cancer-related gene expression pattern stained withcurcumin; the section (b) shows an image of a ras-family cancer-relatedgene expression pattern stained with phloxine; and the section (c) is an(a)+(b) superimposed image.

FIG. 2B shows images of a cancer-related gene expression pattern ofliving cells associated with a specimen 2, and the images are shown inthe same manner as those in FIG. 2A.

FIG. 2C shows images of a cancer-related gene expression pattern ofliving cells associated with a specimen 3, and the images are shown inthe same manner as those in FIG. 2A.

FIG. 3 shows images of a group of healthy living cells associated with aspecimen 4.

FIG. 4A shows three panels (a), (b), and (c) in the upper row of showingthe result of operation of double-staining the inner wall of a largeintestine of a digestive tract of a laboratory mouse with a staincontaining Acid Red and a stain containing a curcumin-based compound inliving body staining, capturing cell images at the surface of the innerwall of the digestive tract and in the interior of the living body overthe range from the surface to a depth of about 30 μm with a confocallaser microscope, and digitizing the images. The section (a) shows animage of the cells stained in living body staining using the staincontaining Acid Red. The section (b) shows an image of the cells stainedin living body staining using the stain containing a curcumin-basedcompound. The section (c) is an (a)+(b) superimposed image. The threepanels (d), (e), and (f) in the lower row show images of the same siteof the large intestine of the digestive tract of the laboratory mousestained in the same living body staining as that in the upper row, fixedwith formalin, and then imaged in the fluorescence antibody technique.The section (d) shows that intracellular actin filaments are visualizedwith Alexa-488-labeled phalloidin. The section (e) shows that theintracellular actin filaments are stained with anti-STAT3 antibody andAlexa-594-labeled secondary antibody at the same time so that thedistribution of a cancer-related gene product STAT3 is shown. Thesection (f) is a (d)+(e) superimposed image.

FIG. 4B shows images of an ultra-early-cancer cell group of healthylarge intestine mucosa of a laboratory mouse stained with curcumin inliving body staining and imaged with a confocal laser microscope.

FIG. 4C shows an image of a sample of human stomach adenoma immediatelyafter surgical excision, stained with curcumin in living body staining,and imaged with a multiphoton laser microscope, with a color regionstained with the curcumin dye extracted.

FIG. 4D shows an image of a sample of the human stomach adenomaimmediately after surgical excision, double-stained with curcumin andAcid Red in living body staining, and imaged under the multiphoton lasermicroscope.

FIG. 4E shows images of the inner wall of the digestive tractdouble-stained with the stain containing curcumin and the staincontaining Acid Red and then imaged under the multiphoton lasermicroscope. The section (a) shows an image of a healthy digestive tract,and the section (b) shows an image of cancer in the ultra-early stage.

FIG. 5A is a diagrammatic view showing the arrangement of the cells ofthe large intestine, which is an example of a digestive tract.

FIG. 5B diagrammatically shows cancer cells in ultra-early cancer thatdevelop in the digestive tract.

FIG. 5C is a diagrammatic view showing that the inner wall of thedigestive tract is imaged under the multiphoton laser microscope and theconfocal laser microscope and further showing examples of cell imagescaptured (a) at the focal plane that coincides with the mucosa surfaceand (b) at the focal plane that is located at the depth of about 50 μmbelow the mucosa surface.

FIG. 6A shows a case where two pattern types, a cancer-related geneSTAT3 expression pattern stained with curcumin in living body stainingand a ras-family cancer-related gene expression pattern stained withphloxine in living body staining, of a lesion (circular structure seenin central portion) called ACF (atypical crypt foci), which isconsidered as one form of a pre-cancer state, are simultaneouslyanalyzed by using images produced under the multiphoton lasermicroscope. The section (a) shows an image of the STAT3 cancer-relatedgene expression pattern stained with curcumin in living body staining.The section (b) shows an image of the ras-family cancer-related geneexpression pattern stained with phloxine in living body staining. Thesection (c) is an (a)+(b) superimposed image.

FIG. 6B is an enlarged view of FIG. 6A and shows the case where the twopattern types, the cancer-related gene STAT3 expression pattern stainedwith curcumin in living body staining and the ras-family cancer-relatedgene expression pattern stained with phloxine in living body staining,of the lesion (circular structure seen in central portion) called ACF(atypical crypt foci), which is considered as one form of the pre-cancerstate, are simultaneously analyzed by using images produced under themultiphoton laser microscope. The section (a) shows an image of theSTAT3 cancer-related gene expression pattern stained with curcumin inliving body staining. The section (b) shows an image of the ras-familycancer-related gene expression pattern stained with phloxine in livingbody staining. The section (c) is an (a)+(b) superimposed image.

FIG. 7 shows a state in which an insertion tube has been inserted into adigestive tract. The section (a) of FIG. 7 shows a state immediatelyafter the insertion tube is inserted, and the section (b) of FIG. 7shows a state in which a space is formed in the digestive tract.

FIG. 8 shows an example of an application unit of a cancer test deviceaccording to a first embodiment.

FIG. 9 The section (a) of FIG. 9 shows that the cancer test deviceaccording to the first embodiment is used to planarize the inner wall ofa digestive tract, and the section (b) of FIG. 9 is a diagrammatic viewshowing a front-end-side end portion of the cancer test device.

FIG. 10 is a schematic view showing the structure of a front end portionof an endoscope in the cancer test device according to the firstembodiment.

FIG. 11 is a block diagram showing the control configuration of thecancer test device according to the first embodiment.

FIG. 12 is a flowchart showing an example of the action of the cancertest device according to the first embodiment.

FIG. 13 is a block diagram showing the control configuration of a cancertest device according to a second embodiment.

FIG. 14 is a diagrammatic view showing the cancer test device accordingto the second embodiment.

FIG. 15 is a schematic view showing a front-end-side end portion of anendoscope of a cancer test device according to a third embodiment.

FIG. 16 is a schematic view showing the entire endoscope.

FIG. 17 is a block diagram showing the control configuration of thecancer test device.

FIG. 18 is a flowchart showing an example of the action of the cancertest device according to the third embodiment.

FIG. 19A is a merged image of the inner wall of the digestive tractstained with the stain containing curcumin and the stain containing AcidRed.

FIG. 19B is the merged image of the inner wall of the digestive tractstained with the stain containing curcumin and the stain containing AcidRed and shows the positional relationship between an imaging axis andthe position in the developed image.

FIG. 20A shows three-dimensional data images illustrating a cell formover a predetermined depth range below the inner wall surface (mucosasurface) and representing an extracted color region stained both withthe curcumin dye and the Acid Red dye.

FIG. 20B shows images representing a color region stained with thecurcumin dye and extracted from the image shown in FIG. 20A.

FIG. 20C shows images representing a color region stained with the AcidRed dye and extracted from the image shown in FIG. 20A.

DESCRIPTION OF EMBODIMENTS

(Finding 1 on which Present Invention is Based)

The present invention is based on findings 1 and 2. Out of the findings,the finding 1 on which the present invention is based and a primaryconfiguration of the invention associated with the finding 1 will firstbe described.

The cancerization mechanism, in which a healthy living cell transformsinto a cancerous cell and the cancerous cell then grows, will first bedescribed. FIG. 1A is a diagrammatic view showing a state in whichcancer-related gene products in a living cell act normally. In the cell,there is a bidirectional signal transduction system that controls celldivision and growth in the positive and negative directions. That is,there are a growth-promoting signal having the function of acceleratingcell growth and a growth-inhibiting signal system having the function ofinhibiting the growth. FIG. 1B is a diagrammatic view showing a state inwhich the cancer-related gene products in the living cell actabnormally. The cross star marks each indicate cancerous mutation thatoccurs in the corresponding cancer-related gene product.

A living cell is formed of a nucleus containing a cell growth gene and acytoplasm that surrounds the nucleus. A cell contains a plurality oftypes of cancer-related genes each formed of proteins. Thecancer-related genes are classified into those that belong to the ras(rat sarcoma) system and the STAT3 (signal transducer and activator oftranscription 3) system, which each transmit the growth-promotingsignal, and the APC (antigen presenting cell)/β-catenin family and thep53 (protein 53) system, which each transmit the growth-inhibitingsignal, as shown in FIG. 1A. That is, the ras-family and STAT3-familycancer-related gene products are classified as gene products in theacceleration system, which transmits a cell growth promoting signal, andthe APC/β-catenin-family and p53-family cancer-related gene areclassified as gene products in the brake system, which transmits a cellgrowth inhibiting signal.

The mechanism that controls cell division and growth will be described.First, when a cell growth factor (EGF), which is an extracellular growthcontrol substance, binds to a receptor located on the cell membrane of aliving cell, the cancer-related gene products of the ras family and theSTAT3 family, which belong to the accelerator system, are activated.When the resultant growth promoting signals are transmitted to thenucleus, a gene group necessary for cell growth is activated in thenucleus. On the other hand, in the cell, the cancer-related geneproducts of the APC/β-catenin family and the p53 family, which belong tothe brake system, are always activated to some extent, and the resultantgrowth inhibiting signals inhibit the activation of the cell growth genein the nucleus to attempt to inhibit the cell growth. In the case wherethe cancer-related genes in the living cell act normally, the effect ofpromoting the cell growth and the effect of inhibiting the cell growthwork in a well-balanced manner, whereby the cells in the living bodygrow adequately.

In contrast, in the case where the ras-family or STAT3-familycancer-related gene product acts abnormally, the cancer-related geneproduct of the ras family or the STAT3 family, which belongs to thegrowth promoting signal system, has higher activity, so that the cellgrowth is enhanced more than necessary, as shown in FIG. 1B. Further,for example, in the case where the APC/β-catenin-family or p53-familycancer-related gene product acts abnormally, the cancer-related geneproduct of the APC/β-catenin family or the p53 family, which belongs tothe growth inhibiting signal system, has lower activity, so that thefunction of inhibiting the cell growth decreases. As described above,when any of the plurality of types of cancer-related gene actsdifferently or abnormally, adequate growth in a healthy cell fails, andenhancement of abnormal cell growth, such as that in a cancer cell,starts.

FIG. 1C is a diagrammatic view showing a stepwise cancerization processof a living cell group on the inner wall surface of a digestive tract.In FIG. 1C, the cancerization process of the living cell group isdivided into a first stage, a second stage, a third stage, and a fourthstage, which are sequentially shown.

The first stage is a stage in which cancerization is about to start inpart of the living cell group. The first stage is believed to occur whenthe degree of activity of the APC/β-catenin-family cancer-related genelowers and the function of inhibiting cell growth decreases accordingly.In this stage, it is shown that the cell growth is slightly enhanced,and that a pre-cancer state, which eventually may transform to a cancercell, has at least occurred.

The second stage is another pre-cancer state in which the cancerizationhas advanced beyond the first stage. In the second stage, it is believedthat the activity of the ras-family cancer-related gene rises so thatthe cell growth has been enhanced. It is further believed theSTAT3-family cancer-related gene is likely to be activated in thisstage. The cancer cell population has a small size having a diameter,for example, greater than or equal to 0.1 mm and smaller than or equalto 0.4 mm. The diameter of the cancer cell population is the diameter ofa circle having the same area as the area of the cancer cell population.This stage is not a stage that immediately endangers the life of thepatient, but it is desirably to lay a treatment plan or otherwise takemeasures in preparation for the future.

ACF (atypical crypt foci), which will be described later and isconsidered as one form of the pre-cancer state, is believed to bedefined by the special name in correspondence with a cell group thatbelongs to the first and second stages and has clear morphologicalfeatures characterized in that the opening or lumen of a gland, whichnormally has a circular shape, has an elongated slit-like shape and thenumber of goblet cells among the glandular cells decreases as comparedwith the number in the normal state.

The third stage is a stage in which part of the living cell group isinfiltrated and cancer cells manifest. The third stage is believed tooccur when the activity of the p53-family cancer-related gene lowers sothat the function of inhibiting the cell growth decreases. In thisstage, in which the activity of brake-system cancer-related geneproducts of both the p53-system and the APC/β-catenin-family lowers sothat the function of inhibiting the cell growth greatly decreases, thegrowth of the cancer cells progresses at an accelerated tempo, and thecancer cells infiltrate the surrounding tissue. When the third stage isreached, the diameter of the portion in the third stage reaches 0.5 mmor greater, and if the third stage is left with no measure taken, cancerthat causes the death of the individual is completed.

The fourth stage is a stage in which after the cancer cells completed inthe third stage transform to cancer, further gene mutation occurs, andthe cancer has progressed to malignant cancer, which is likely tofurther cause cell growth, infiltration, and metastasis. This stage is astage in which the cancer metastasizes to remote organs other than thedigestive tract and is a dangerous stage that endangers the life of thepatient. It is believed that the speed at which the first stageprogresses to the fourth stage depends on the state of the activity ofthe cancer-related genes.

FIG. 1D shows an example of a human cancer cell growth curve.

The number of cancer cells typically increases in accordance with apredetermined growth curve, as shown in FIG. 1D. For example, the growthcurve has a small gradient for three years corresponding to the stage inwhich cancerization is about to start (period for which diameter ofcancer cell population is smaller than 0.2 mm), whereas the gradient ofthe growth curve increases after four years and later (period for whichdiameter of cancer cell population is greater than or equal to 0.5 mm).The gradient of the growth curve then decreases after seven years andlater. In general, the timing when cancer is clinically found andtreated is after seven years or later. The reason for this is that nocancer cell population can be detected unless the diameter thereofincreased to a value greater than or equal to 10 mm.

It is a noteworthy fact that the number of cells exponentially increasesover the range of the growth curve indicated by the broken line A. Theexponential increase means that the cancerous gene mutation that shouldoccur in the first to third stages of the cancer cells has completed andthe cancer cells repeatedly divide at a fixed uniform speed. If thecancer cell population can be detected at an early stage of theexponential increase (ultra-early cancer), that is, at a stage in whichabnormality occurs in the cancer-related gene expression patterns butthe cancer cell population itself has a small diameter of 1 mm orsmaller, the cancer can be permanently cured because the ultra-earlycancerous site is sufficiently small and can be readily fully removed.As described above, if the grade of the cancerization can be grasped inthe form of abnormality in a cancer-related gene expression pattern inthe ultra-early stage, the cancer can be permanently cured before thecancer develops to a dangerous stage.

Under the background described above, the present inventors haveattempted to grasp the grade of cancerization by imaging acancer-related gene expression pattern of a living cell group with amultiphoton laser microscope or a confocal laser microscope andvisualizing the state of the activity of the cancer-related gene.

To visualize a cancer-related gene expression pattern of a living cellthe present inventors have used a stain containing an edible dye tostain a cancer-related gene product in a chromatic color and imaged thestained cancer-related gene product. An edible dye is a natural dye oran artificial dye that is allowed to be administered to human (foodcoloring dye or dye that can be administered in the form of supplement,for example).

Specifically, a stain containing a curcumin-based compound (curcumin,C₂₁H₂₀O₆) was prepared as the stain that selectively stains theSTAT3-family cancer-related gene product. Further, a stain containingphloxine (C₂₀H₂Br₄Cl₄Na₂O₅) was prepared as the stain that selectivelystains the ras-family cancer-related gene expression pattern.

More specifically, a curcumin containing solution containing 1 wt % ofcurcumin was prepared as the stain containing a curcumin-based compound,and a phloxine containing solution containing 1 wt % of phloxine wasprepared as the stain containing phloxine. The stain containing acurcumin-based compound may instead be a curcumin solution (undilutedliquid is, for example, liquid containing 5%-curcumin, 45%-glycerol, and50%-ethanol) diluted with a physiological saline solution at a ratioranging from 1/5 to 1/100. The stain containing 1%-phloxine may insteadbe a phloxine solution (10 mg/mL of undiluted liquid) with no change orhaving a concentration of 100% or diluted at a ratio of 1/10.

The stain containing a curcumin-based compound was used to stain theexpressed STAT3-family cancer-related gene product in the living cell,and the stained product was then washed with physiological salinesolution for about 10 seconds three times. The stain containing phloxinewas used to stain the ras-family cancer-related gene expression patternin the living cell, and the stained pattern was then washed withphysiological saline solution for about 10 seconds three times. The thusperformed double staining allowed simultaneous analysis of the amountsof expression of the STAT3-family and ras-family cancer-related geneproducts. The staining period for each of the stains ranged from 2 to 5minutes. Since the concentrations described above allow the stains topenetrate into the cytoplasm but does not allow the stains to penetrateinto the nucleus in the cell as long as the period having elapsed sincethe staining started is 10 minutes or shorter, the nucleus surrounded bythe cytoplasm can be sharply visualized, whereby the analysis can bemore distinctly performed.

The expression patterns of the STAT3-family and ras-familycancer-related gene products stained with the stains described abovewere imaged with a multiphoton laser microscope (FV1000MPE manufacturedby Olympus Corporation). The wavelength of the laser light was 840 nm.An imaging target was the inner wall of the large intestine of alaboratory mouse, and the inner wall of the large intestine was soobserved as to be divided into a plurality of sites where cancer cellsdeveloped (specimens 1, 2, and 3) and a healthy cell site where nocancer cell has developed (specimen 4).

FIG. 2A shows images of the cancer-related gene expression pattern of agroup of living cell populations of the laboratory mouse large intestineassociated with the specimen 1: The section (a) shows an image of theSTAT3-family cancer-related gene product with the cancer cells stainedmore heavily than the healthy cells with the stain containing acurcumin-based compound; the section (b) shows an image of theras-family cancer-related gene expression pattern stained with the staincontaining phloxine; and the section (c) is an (a)+(b) superimposedimage. FIG. 2A, and FIGS. 2B and 2C and FIG. 3 , which will be describedlater, are each a monochromatic image but are inherently a color image.

In the section (a) of FIG. 2A, the STAT3-family cancer-related geneproduct is expressed in a green fluorescence color. The ratio of theregion stained in the green fluorescence color to the entire screen,that is, the proportion of the region occupied by cells dominated by theSTAT3-family cancer-related gene expression is 30%.

In the section (b) of FIG. 2A, the ras-family cancer-related gene isexpressed in a red fluorescence color. The ratio of the region stainedin the red fluorescence color to the entire screen, that is, theproportion of the region occupied by cells dominated by ras-familycancer-related gene expression is 80%.

In the section (c) of FIG. 2A, the region where the STAT3-familycancer-related gene product and the ras-family cancer-related geneexpression pattern coexist is expressed in a yellow fluorescence color,the region where only the STAT3-family cancer-related gene expressionexists is expressed in the green fluorescence color, and the regionwhere only the ras-family cancer-related gene expression exists isexpressed in the red fluorescence color. The ratio of the region stainedin the yellow fluorescence color to the entire screen is 10%.

In the group of living cell populations of the laboratory mouse largeintestine associated with the specimen 1, the proportion of the regionoccupied by cells dominated by STAT3-family cancer-related geneexpression was 30%, the proportion of the region occupied by cellsdominated by ras-family cancer-related gene expression was 80%, and theproportion of the region where the two types of cells described abovecoexist was 10%. The state of the group of living cell populationsassociated with the specimen 1 is believed to be the second or thirdstage (ultra-early cancer) shown in FIG. 1C in terms of the grade ofcancerization. That is, the stage of the group of living cellpopulations is diagnosed as follows: Cells that are positive in terms ofboth STAT3-family and ras-family account for 10%; the group of thesecells has advanced beyond at least the second stage; the population ofthese cells has a diameter of at least 0.2 mm (see FIG. 1D); and it istherefore estimated that the group of living cell populations is likelyto have already entered the third stage (ultra-early cancer).

FIG. 2B shows images of the cancer-related gene expression pattern of agroup of living cell populations associated with the specimen 2, and theimages are shown in the same manner as those in FIG. 2A.

In the section (a) of FIG. 2B, the ratio of the region stained in thegreen fluorescence color to the entire screen, that is, the proportionof the region of the STAT3-family cancer-related gene expression is 50%.In the section (b) of FIG. 2B, the ratio of the region stained in thered fluorescence color to the entire screen, that is, the proportion ofthe region of the ras-family cancer-related gene expression is 90%. Inthe section (c) of FIG. 2B, the ratio of the region stained in theyellow fluorescence color to the entire screen, that is, the proportionof the region where the STAT3-family cancer-related gene and theras-family cancer-related gene coexist is 20%. In the group of livingcells, the STAT3-family and ras-family cancer-related genes areactivated, and cancerization that is diverse in terms of gene expressionis recognized, as in the specimen 1 (see FIG. 2A). The stage of thegroup of living cell populations associated with the specimen 2 isdiagnosed as follows: Cells that are positive in terms of bothSTAT3-family and ras-family account for 20%; the group of these cellshas advanced beyond at least the second stage; the population of thesecells has a diameter of at least 0.2 mm (see FIG. 1D); and it istherefore estimated that the group of living cell populations is likelyto have already entered the third stage (ultra-early cancer).

FIG. 2C shows images of the cancer-related gene expression pattern of agroup of living cell populations associated with the specimen 3, and theimages are shown in the same manner as those in FIG. 2A.

In the section (a) of FIG. 2C, the ratio of the region stained in thegreen fluorescence color to the entire screen, that is, the proportionof the region of the STAT3-family cancer-related gene expression is 30%.In the section (b) of FIG. 2C, the ratio of the region stained in thered fluorescence color to the entire screen, that is, the proportion ofthe region of the ras-family cancer-related gene expression is 75%. Inthe section (c) of FIG. 2C, the ratio of the region stained in theyellow fluorescence color to the entire screen, that is, the proportionof the region where the STAT3-family cancer-related gene and theras-family cancer-related gene coexist is 5%. In the group of livingcells associated with the specimen 3, the ras-family cancer-related geneis not activated, so that the gene expression in the cancer cells is notas diverse as that in the specimen 1 (see FIG. 2A).

The stage of the group of living cell populations associated with thespecimen 3 is diagnosed as follows: Cells that are positive in terms ofboth STAT3-family and ras-family account for 5%; the group of thesecells has advanced beyond at least the second stage; and the populationof these cells has a diameter of about 0.1 mm (see FIG. 1D). Inconsideration of the state in which the STAT3-family positive cells arescattered in the form of many island-shaped portions, it is expectedthat the mobility of the cells has been enhanced and the infiltrationhas increased, and it is then estimated that the cells are likely tohave already entered the third stage (ultra-early cancer). The detailedcell-level analysis of the states of the expression of manycancer-related gene products allows cancer stage diagnosis andcancerization grade diagnosis. It is believed that the metastasis isevaluated based on the dispersibility of cancer cells, and that the sizeof a cell population indicates the growth ability.

As shown, for example, in the section (a) of FIG. 2C, a case whereindividual cancer populations per unit area each have a small diameterbut there are a large number of scattered cancer populations is believedto indicate that metastasis advances. As described above, the fact thatmetastatic cancer has a small average size (diameter) can beincorporated in the analysis approach. It is desirable in this case thatthe metastatic level is classified into three to five stages, and that adetermined metastatic level is described along with the grade ofcancerization expressed, for example, by the ras and STAT3 stainedareas.

FIG. 3 shows images of a group of healthy living cells of the largeintestine of the laboratory mouse associated with the specimen 4. In thesections (a) to (c) of FIG. 3 , no green, red, or yellow fluorescencecolor is present in the screen, but the screen is expressed in a darkcolor. In the group of living cells associated with the specimen 4having no cancer cell, the amounts of expressed STAT3-family andras-family cancer-related gene products are not enhanced.

Analysis of expression of cancer-related genes in a group of livingcells as described above allows determination of the grade ofcancerization. That is, according to the primary configuration of thepresent invention, ultra-early cancer having a so small lesion size thata current test device (such as endoscope) cannot detect even thepresence of the ultra-early cancer can be found in an early stage, andthe prognosis of the cancer patient can be understood through evaluationof the grade of the cancer cells. The cancer can be permanently cured byfully removing the cancerous site in an early stage as described above.

FIG. 4B shows images of healthy large intestine mucosa and colorectalcancer stained with curcumin in living body staining and imaged with aconfocal laser microscope and shows that the contour of each cell andthe shape of the nucleus in the cell can be clearly visualized becausecurcumin is loaded into the cytoplasm but is not loaded into the nucleusso that pathological diagnosis can be reliably performed. In the section(b) of FIG. 4B, since crypts cannot be seen due to the colorectal cancersite, unlike the section (a) of FIG. 4B, the section (b) of FIG. 4B canbe used in cancer evaluation.

Further, the section (a) of FIG. 4C shows an image of a surgicallyexcised fresh specimen of human stomach adenoma stained with curcumin exvivo in living body staining and imaged with a multiphoton lasermicroscope. The section (a) of FIG. 4D shows an image of a surgicallyexcised fresh specimen of human stomach adenoma double-stained withcurcumin and Acid Red ex vivo in living body staining. Since the contourof each cell and the shape of the nucleus can be clearly visualized,pathological diagnosis can be reliably performed also in this case. Thegraphs drawn in the right section (b) of FIGS. 4C and 4D show that thehatched bands labeled with Filter1 and Filter2 represent the wavelengthwidths of filters used to measure the fluorescence, that E2 representsthe characteristic of the fluorescence emitted from curcumin, and thatE7 represents the characteristic of the fluorescence emitted from AcidRed. Therefore, in the section (a) of FIG. 4D, the difference inwavelength of the fluorescence provided by the two-color staining isexpressed in the form of the difference in color. FIG. 4D shows amonochromatic image and is therefore considered to show the differencein contrast but is actually captured as an image that is clearlydividable in terms of wavelength. In FIGS. 4C and 4D, the regions whichare labeled with II and where cancerization is about to start are each akind of cytologic atypia, and nuclei in the gland are arranged in tworows and division due to cancer is about to start. That is,visualization of the nuclei allows clear grasp of a situation in whichcancerization is about to start on a cell basis.

The present inventors have ascertained, as an example in whichcancer-related gene expression patterns are selectively stained, that astain containing a curcumin-based compound is used to selectively staina STAT3-family cancer-related gene product in a chromatic color. FIG. 4Ashows monochromatic images that are inherently color images. The imagescan be produced both with a confocal laser microscope and a multiphotonlaser microscope.

The sections (a), (b), and (c) of FIG. 4A are images of a sample that isa double-stained living cell group of the inner wall of the intestine ofa living laboratory mouse produced by staining the inner wall of theintestine with a 1%-curcumin solution and then staining thecurcumin-stained inner wall with 1%-Acid Red (C₂₇H₂₉N₂NaO₇S₂). Aconfocal laser microscope was used as the image device.

In the section (a) of FIG. 4A, the region stained with Acid Red is shownin deep red, so that the structure of capillaries and connective tissue(mesh structure) around the glands of the large intestine is visualized.In the section (b) of FIG. 4A, the region stained with thecurcumin-based compound is shown in deep green. The section (c) of FIG.4A is an (a)+(b) superimposed image. It is believed based on the section(c) of FIG. 4A that ultra-early cancer has developed in the regionheavily stained with the curcumin-based compound because the structureof the glands and the capillaries and connective tissue around theglands (crypt structure) stained with Acid Red disappears and isdisturbed.

The three panels in the upper row of FIG. 4A show that (b) there is acell group which is enclosed by the white-line rectangle and where theliving body staining using the stain containing a curcumin-basedcompound stained cancer cells more heavily than healthy cells, and thatcomparison of the enclosed site with (a) the same site of the image ofthe cell group stained in the living body staining using the staincontaining Acid Red shows that the mesh pattern (crypt pattern) in whichthe capillaries stained with Acid Red surround the glands disappears. Itis therefore determined that the site containing the cell group heavilystained with the curcumin stain is ultra-early cancer. The three panels(d), (e), and (f) in the lower row of FIG. 4A show images of the samesite of the large intestine of the digestive tract of the laboratorymouse imaged in the same living body staining as that in the upper row,fixed with formalin, and then imaged in the fluorescence antibodytechnique. The section (d) shows that intracellular actin filaments werevisualized with Alexa-488-labeled phalloidin. The section (e) shows thatthe intracellular actin filaments were stained with anti-STAT3 antibodyand Alexa-594-labeled secondary antibody at the same time so that thedistribution of the cancer-related gene product STAT3 is shown. Thesection (f) is a (d)+(e) superimposed image.

The sections (d), (e), and (f) of FIG. 4A are produced by fixing theintestine of the laboratory mouse described above with formalin, thenimmunostaining the intestine with an anti-STAT3 antibody that bonds tothe STAT3-family cancer-related gene product, and imaging the samelocation as those surrounded by the white frames in the sections (a),(b), and (c). In the sections (d) to (f), the samples were fixed withformalin and therefore slightly shrunk.

The three panels in the lower row were obtained by imaging the whiterectangular sites in the upper three panels. The distribution of thecell group shown in the section (b) in the upper row and heavily stainedwith curcumin in the living body staining coincides with thedistribution of the cell group shown in the section (e) in the lower rowand having the cancer-related gene product STAT3 highly expressed, whichsuggests that living body staining of the living cells with curcumindetects the cancer-related gene product STAT3.

Further, out of the three panels in the lower row, comparison betweenthe distribution of the cell group shown in the section (e) and havingthe cancer-related gene product STAT3 highly expressed and thedistribution of the intracellular actin filaments in the section (d)indicates that the cell group in which STAT3 is highly expressed has asparse distribution of the actin filaments, which indicates that theinter-cell bonding is sparse. Based on the fact that the inter-cellbonding is typically sparse in a cancerous cell, the cell group in whichSTAT3 is highly expressed is formed of cancerous cells.

In summary, the three panels in the lower row prove that cancerizationhas occurred in the central-part cell group that is poor in actinfilaments, and that the cell group coincides with the cell group inwhich the cancer-related gene product STAT3 is highly expressed.Further, the three panels in the upper row prove that the cell group inwhich the cancer-related gene product STAT3 is highly expressed isformed of ultra-early cancer cells more heavily stained in the curcuminliving body staining than the healthy cells.

These images were captured with a confocal laser microscope, and similarimages can be captured with a multiphoton laser microscope.

The section (d) of FIG. 4A primarily shows the contours of the cellsvisualized by actin fluorescence. The section (e) of FIG. 4A shows cellsimmunostained with the anti-STAT3 antibody. The section (f) of FIG. 4Ais the (d)+(e) superimposed image and shows that the amount of whiteactin reaction decreases in the island-shaped portion in a centralportion of the screen, and that the amount of green STAT3-family proteinimmunoreaction increases. It is therefore believed that a large amountof STAT3-family cancer-related gene product has been expressed in theisland-shaped portion.

Referring to the images in FIG. 4A, since the region in the section (c)of FIG. 4A, where cancer cells have developed, coincides with the regionin the section (f) of FIG. 4A, where STAT3-family cancer-related geneproduct is detected, it is indicated that ultra-early cancer hasdeveloped and the cancer-related gene product relating to theultra-early cancer has been expressed in the region heavily stained withthe curcumin-based compound in the living cells. As described above,differences in luminance and fluorescence color or a difference ingeometric pattern allows image-based detection of expression of acancer-related gene in an ultra-early stage and analysis of the grade ofcancerization in computer-based automatic diagnosis. The automaticdiagnosis employs, for example, a method for identifying the expressionlocation based on highly probable luminance and analyzing a regulardistribution of the crypt structure to determine the grade ofcancerization based on the degree of disappearance of the structure andthe state of mixed fluorescence colors. In a method using a geometricpattern, the pattern varies on an organ basis. In the example in thesection (c) of FIG. 4A, it is conceivable to employ, for example, amethod for counting the number of dark portions in a fixed area of a500-μm square (dark hole pattern) to quantify the density of the darkportions. The automatic diagnostic can further be useful forartificial-intelligence-based diagnosis for recognition from storedimages of a large number of cancer-related gene expression patterns andhealthy cell patterns. In addition to the hole pattern of the cryptstructure, depending not only on the number of repetitions of dark andbright portions but the depth of penetration of a living body stain intoa living cell, the depth of imaging and the wavelength in confocal andmultiphoton laser microscopy, a difference between stains, and otherfactors, the geometric pattern is imaged as a different pattern in somecases, for example, in the sections (a) and (b) of FIG. 4E, which showimages of stained intestine of a laboratory mouse. Measurement of thedensity of the island pattern based on the glands and capillaries,recognition of disturbance of the island pattern, for example, in thesection (a) of FIG. 4E, and any other diagnosis method can be employed.Further, distance measurement, such as the interval between the darkportions, the diameters of the dark portions, and other factors in thesection (c) of FIG. 4A and the interval in the island pattern, thediameter of the island pattern, and other factors in the section (a) ofFIG. 4E, is useful in evaluation of the disturbance of the pattern.

As described above, detailed cell-level analysis of the states ofexpression of a large number of cancer-related gene products allowscancer stage diagnosis and cancerization grade diagnosis. It is believedthat the metastasis is evaluated based on the dispersibility of cancercells, and that the size of a cell population indicates the growthability.

For example, a case where individual cancer populations per unit areaeach have a small diameter but there are a large number of scatteredisland-shaped populations is believed to indicate that metastasisadvances. As described above, the fact that metastatic cancer has asmall average size can be incorporated in the analysis approach.

(Finding 2 on which Present Invention is Based)

The finding 2, on which the present invention is based, and a primaryconfiguration of the invention associated with the finding 2 will nextbe described.

The relationship between the internal structure of a living body andcancer cells will first be described.

The interior of a living body contains the digestive tract, therespiratory system, the renal/urinary system, the utero-ovarianreproductive system, and other organs, the cerebrospinal nervous system,and other body sites. Examples of the digestive tract may include theesophagus, the stomach, the small intestine, and the large intestine.

FIG. 5A is a diagrammatic view showing the arrangement of the cells ofthe large intestine, which is an example of a digestive tract 112. Forexample, the inner wall of the large intestine is formed of a gland 130,which secretes mucus, and an epithelium 120, which is located in aportion closer to an inner wall surface (mucosa surface) 113 than thegland 130 and absorbs water when coming into contact with food. Theepithelium 120 is formed of a plurality of epithelial cells 121 arrangedalong the inner wall surface 113. The epithelial cells 121 each have anucleus 125 and cytoplasm 126. The gland 130 is so shaped that part ofthe epithelium 120 is recessed in the form of a pot. The gland 130 isformed of a plurality of glandular cells 131, and the glandular cells131 each have a nucleus 135 and cytoplasm 136. The recessed portions ofthe gland 130 are called crypts 138 of the gland 130. A basementmembrane 137, capillaries 132, and connective tissue 133 are formed inthe portion inside the epithelial cells 121 and the portion around theglandular cells 131. A thin layer of the mucus secreted from the gland130 is formed on the surfaces of the epithelial cells 121, and theepithelial cells 121 are protected by the mucus layer.

FIG. 5B diagrammatically shows a cancer cell population 152, whichdevelops in the digestive tract 112. The ultra-early-stage cancer cellpopulation 152, which develops in the digestive tract 112, is generallybelieved to develop in a position below the inner wall surface (mucosasurface) 113 of the digestive tract 112 at a depth of about 1 mm orsmaller. If the early-stage cancer cell population 152, which has notyet reached and penetrated into a muscular layer of mucosa 160, can befound over a wide range of the mucosa of the digestive tract with nomissing cancer cell population, the number of conditions leading to anadvanced cancer, which is a state in which the cancer cell populationproliferates beyond the muscular layer of mucosa 160 and spreads toanother organ, can be reduced.

FIG. 5C is a diagrammatic view showing that the inner wall of thedigestive tract 112 is imaged under a multiphoton laser microscope and aconfocal laser microscope. To irradiate the inner wall of the digestivetract 112, which is an imaging target, with laser light L, an objectivelens 16 of the multiphoton laser microscope and the confocal lasermicroscope is so disposed as to face the inner wall surface 113 of thedigestive tract 112, as shown in FIG. 5C. FIG. 5C is a diagrammatic viewwith the left half of FIG. 5C showing that the inner wall of thedigestive tract is imaged under the multiphoton laser microscope or theconfocal laser microscope and the right half of FIG. 5C showing examplesof cell images captured (a) at the focal plane that is the plane of thecross section taken along the line a-a and that coincides with themucosa surface and (b) at the focal plane that is the plane of the crosssection taken along the line b-b and that is located at the depth ofabout 50 μm below the mucosa surface. The multiphoton laser microscopeallows imaging over the range from the mucosa surface to a depth ofabout 500 μm or a depth of 1000 μm, and the confocal laser microscopeallows imaging over the range from the mucosa surface to a depth ofabout 50 μm or a depth of 100 μm.

To primarily image the epithelial cells 121, the objective lens 16 is sodisposed that the focal point of the objective lens 16 coincides withthe inner wall surface (mucosa surface) 113. As a result, the epithelialcells 121 and other parts are imaged as shown in the section (a) of FIG.5C, which is a diagrammatic view taken along the line a-a in FIG. 5C. Onthe other hand, to primarily image the glandular cells 131, thecapillaries 132, and the connective tissue 133, the objective lens 16 isso disposed that the focal point of the objective lens 16 coincides witha position below the inner wall surface (mucosa surface) 113 at a depthof 10 μm or greater. As a result, the glandular cells 131, thecapillaries 132, and the connective tissue 133 are imaged as shown inthe section (b) of FIG. 5C, which is a diagrammatic view taken along theline b-b in FIG. 5C.

For example, if the size and shape of a cancer-related gene expressionpattern that appears in the nucleus 125 or 135 or a crypt 138 of thegland 130 can be detected in the pre-cancer state (second stage shown inFIG. 1C), the grade of cancerization can be determined in theultra-early stage, in which the diameter of the cancer cell populationis greater than or equal to 0.5 mm but smaller than or equal to 1 mm.

FIG. 6A shows images of a cancer-related gene expression pattern ofliving cells. FIG. 6B is an enlarged view of FIG. 6A. FIGS. 6A and 6Bshow monochromatic images that are inherently color images.

FIG. 6A shows a case where two pattern types, a cancer-related geneSTAT3 expression pattern stained with curcumin in living body stainingand a ras-family cancer related gene expression pattern stained withphloxine in living body staining, of a lesion (circular structure seenin central portion) called ACF (atypical crypt foci), which isconsidered as one form of the pre-cancer state, are simultaneouslyanalyzed by using the images produced under the multiphoton lasermicroscope. The section (a) shows an image of the STAT3 cancer-relatedgene expression pattern stained with curcumin in living body staining.The section (b) shows an image of the ras-family cancer-related geneexpression pattern stained with phloxine in living body staining. Thesection (c) is an (a)+(b) superimposed image.

FIGS. 6A and 6B show images produced by actually imaging thecancer-related gene expression patterns stained with a stain 45 underthe multiphoton laser microscope (FV1000MPE manufactured by OlympusCorporation). The wavelength of the laser light was 840 nm, and theimaging target was a laboratory mouse. The stain is the same as thestain shown in the finding 1, the stain containing a curcumin-basedcompound (curcumin, C₂₁H₂₀O₆) as the stain that selectively stains theSTAT3-family cancer-related gene product, and the stain containingphloxine (C₂₀H₂Br₄Cl₄Na₂O₅) as the stain that selectively stains theras-family cancer-related gene expression pattern.

FIGS. 6A and 6B each show a state in which part of the crypts 138 hasbeen stained in green and the STAT3-family cancer-related gene producthas been expressed in the crypts 138. The structure of the centralportion of FIGS. 6A and 6B is called ACF (atypical crypt foci)pre-cancer state. The structure of the central portion in FIGS. 6A and6B forms a gland-like structure in which glandular cells are arranged.The opening or lumen of the gland in the central portion has a circularshape in the healthy colorectal mucosa, whereas the structure of thecentral portion in FIGS. 6A and 6B has an elongated slit-shaped opening,and the number of goblet cells among the glandular cells decreases thanthat under the healthy condition. The structure of the central portiontherefore has obvious morphological characteristics of ACF. In thepre-cancer state ACF, (a) slight enhancement of the STAT3 cancer-relatedgene expression stained with curcumin in living body staining and (b)moderate enhancement of ras-family cancer-related gene expressionstained with phloxine in living body staining are recognized. Further,the crypts 138 in the healthy state each have a roughly circular shape,whereas in the ACF pre-cancer state shown in FIGS. 6A and 6B, two crypts138 adjacent to each other each have an elongated deformed shape, fromwhich it can be determined that the living cells are in an abnormalstate. As described above, detecting the size and shape of eachcancer-related gene expression pattern in the pre-cancer state allowsdetermination of the grade of cancerization in the early stage andunderstanding of the prognosis of the cancer patient.

First Embodiment

A first embodiment will be described below in detail with reference tothe drawings.

The embodiments described below are each a preferable specific exampleof the present invention. Numerical values, shapes, materials,constituent elements, the positions where the constituent elements arearranged, the form in accordance with which the constituent elements areconnected to each other, steps, the order of the steps, and otherfactors shown in the following embodiments are presented by way ofexample and are not intended to limit the present invention. The presentinvention is specified by the claims. Therefore, out of the constituentelements in the following embodiments, a constituent element that is notdescribed in any independent claim will be described as an arbitraryconstituent element. Further, in the drawings, substantially the sameconfigurations have the same reference character, and a duplicateddescription of such configurations will be omitted or simplified.

[1. Overall Configuration of Cancer Test Device]

A cancer test device according to the present embodiment is a devicecapable of finding cancer cells having developed in the digestive tract,the respiratory system, the renal/urinary system, the utero-ovarianreproductive system, the cerebrospinal nervous system, and other bodysites in an early stage of the cancer. The cancer test device can notonly perform the cancer test but treat the cancer cells having developedin the living body. Further, the cancer test device can handle not onlythe interior of a living body but a fresh ex-vivo sample within about 20minutes immediately after the sample is surgically excised with thesample kept intact and can pathologically diagnose cancer and analyzethe expression of the cancer-related genes in the same method as thatused for the interior of the living body. For example, a sample needs tobe frozen, sliced into a thin specimen, and stained with hematoxylin andeosin (HE) in related art, so that whether or not cancer cells arepresent in a resection stump is pathologically diagnosed and requires atleast 20 minutes. On the other hand, the approach according to thepresent embodiment allows accurate diagnosis with no missing lesion in 3to 5 minutes (rapid intraoperative diagnosis). Further, the cancer testdevice and test method according to the present invention are applicableto even an excised, cultured-state ips cell, an ES cell, a MUSE cell, orany other cell other than a living body.

Further, the cancer test device according to the present embodiment isan endoscope-shaped test device. The following description will be madewith reference to a case where the digestive tract in a living body istested.

[1.1. Configuration for Test Preparation]

The configuration of the cancer test device for test preparation willfirst be described.

Since the inner wall of an actual digestive tract has irregularities, itis desirable to widen the digestive tract to make the digestive tractimageable before the inner wall is imaged by using the cancer testdevice. To this end, the cancer test device according to the presentembodiment includes an insertion tube that widens the digestive tract.

FIG. 7 shows a state in which an insertion tube 20 has been insertedinto the digestive tract 112. The section (a) of FIG. 7 shows a stateimmediately after the insertion tube 20 is inserted, and the section (b)of FIG. 7 shows a state in which a space S is formed in the digestivetract 112.

The insertion tube 20 has a supply port 42, through which fluid issupplied, and a recovery port 43, through which the supplied fluid isrecovered, as shown in the section (a) of FIG. 7 . The insertion tube 20is further provided with a first balloon 21 and a second balloon 22. Thefirst balloon 21 and the second balloon 22 bulge and shrink when thefluid (gas or liquid) is injected into and discharged from the balloons21 and 22. The first balloon 21 is provided in a position shifted fromthe supply port 42 toward the front end of the insertion tube 20, andthe second balloon 22 is provided in a position shifted from therecovery port 43 toward the rear side of the insertion tube 20 (oppositethe front end). Causing the first balloon 21 and the second balloon 22to bulge in the digestive tract 112 creates a closed space S sandwichedbetween the first balloon 21 and the second balloon 22, as shown in thesection (b) of FIG. 7 .

[1.2. Basic Configuration of Cancer Test Device]

The basic configuration of a cancer test device 1 will next be describedwith reference to FIGS. 8 to 11 . FIG. 8 shows an example of anapplication unit 40 of the cancer test device 1.

The cancer test device 1 according to the present embodiment includesthe application unit 40, which applies a stain 45 onto the inner wall ofthe digestive tract 112 in the closed space S.

The cancer test device 1 supplies the stain 45 from the application unit40, which contains the stain 45, via the insertion tube 20 and thesupply port 42 into the space S to apply the stain 45 onto the innerwall of the digestive tract 112, as shown in FIG. 8 . Cancer-relatedgene products in living cells of the digestive tract 112 are stainedwith the applied stain 45 in a chromatic color.

The stain 45 may, for example, be a single stain formed of a staincontaining a curcumin-based compound or a stain containing phloxine. Itis, however, desirable to use two stains, the stain containing acurcumin-based compound and the stain containing phloxine. Using thestain 45 containing a curcumin-based compound allows grasp of the stateof a STAT3-family cancer-related gene expression pattern, and using thestain 45 containing phloxine allows grasp of the expression state of aras-family cancer-related gene product.

Curcumin-based compounds include not only curcumin, of course, buthighly water-soluble curcuminoid (mixture of several types of curcuminderivatives).

The stain that stains the ras-family cancer-related gene expressionpattern can, in place of phloxine described above, be a stain containingany of the following materials:

erythrosine (C₂₀H₈I₄O₅)

merbromin (C₂₀H₈Br₂HgNa₂O₆)

fast green FCF (C₃₇H₃₄N₂Na₂O₁₀S₃)

meclocycline sulfosalicylate (C₂₉H₂₇ClN₂O₁₄S)

Before the staining, the interior of the digestive tract 112 may becleaned and the mucus in the digestive tract 112 may be removed via thesupply port 42 and the recovery port 43.

The section (a) of FIG. 9 shows that the cancer test device 1 is used toplanarize the inner wall of the digestive tract 112.

After the stain 45 is applied to a cell group in the living body, gasis, for example, supplied via the supply port 42 to cause the digestivetract 112 to bulge, as shown in the section (a) of FIG. 9 . As a result,the inner wall of the digestive tract 112 is stretched and planarized.It is desirable that the irregularities of the planarized inner wallsurface 113 have a height difference, for example, smaller than or equalto 0.2 mm. Planarizing the inner wall of the digestive tract 112 allowsprecise grasp of the states of the inner wall surface 113 and thecancer-related genes in the living cell group in a position below theinner wall surface 113 at a predetermined depth.

The section (b) of FIG. 9 is a schematic view showing a front-end-sideend portion of an endoscope 2 of the cancer test device 1 in FIG. 11 .FIG. 10 is a schematic view showing the structure of a rotary portion atthe front end of the endoscope 2. FIG. 11 is a block diagram showing thecontrol configuration of the cancer test device 1.

The cancer test device 1 includes, in addition to the application unit40 described above, an imaging unit 10 including the endoscope 2, and acontrol unit 50, which controls the motion of the imaging unit 10 andthe application unit 40. The control unit 50 includes an evaluation unit52, which evaluates the grade of cancerization, and a storage unit 51,which stores information that serves as an evaluation reference when thegrade of cancerization is evaluated. The evaluation unit 52 and thestorage unit 51 will be described later.

The cancer test device 1 further includes a laser oscillator 60, anoptical part 65, and an image processing unit 70.

Laser light L emitted from the laser oscillator 60 is reflected off adichroic mirror 66, which is the optical part 65, further reflected offa mirror 19 in the endoscope 2, and applied to the living body. Thecancer-related gene products in the living cells irradiated with thelaser light L produce fluorescence, and the fluorescence is reflectedoff the mirror 19, passes through the dichroic mirror 66, and isdetected with a photodetector 35. The light detected with thephotodetector 35 is converted into an electric signal, and the imageprocessing unit 70 forms an image according to the electric signal. Atwo-dimensional scanner 67 is built in the space between the laseroscillator 60 and the dichroic mirror 66 (FIG. 11 ). The two-dimensionalscanner 67 scans a fixed area of an imaging target region with theradiated laser light in the X-Y directions to allow the laser light,which acts as a point, to form a planar image. Since the color of thefluorescence changes in accordance with the stain, the photodetector 35is formed of a plurality of photodetectors, and a color separationoptical filter is disposed on the upstream side of the photodetector 35for color separation. The color may instead be separated by using a CMOS(complementary metal oxide semiconductor) device or a CCD (chargecoupled device) as the photodetector 35.

The laser oscillator 60 to be used in the multiphoton laser microscopeis configured to have a pulse width ranging from several tens to severalhundreds of femtosecond and a pulse frequency ranging from several tensto several hundreds of megahertz. The laser light L in the presentembodiment is two-photon laser light, which is a kind of multiphotonlaser light, and the laser oscillator 60 uses, for example, a pulselaser capable of emitting light having a wavelength of 800 nm and apower of 3.2 W at the maximum. In the imaging operation, the laseroutputs laser light having a power ranging from 0.16 to 0.32 W. Settingthe wavelength at 800 nm or longer can prevent photons that belong tothe ultraviolet region (wavelength shorter than 400 nm) from beingproduced in the half-wavelength light produced in the multiphotonexcitation process. The laser oscillator 60 to be used in the confocallaser microscope is, for example, a continuous-wave (CW) laseroutputting light having a visible wavelength for a typical confocallaser microscope.

The dichroic mirror 66, which is the optical part 65, reflects lighthaving the same wavelength as that of the laser light L and transmitslight having the other wavelengths. The laser light L emitted from thelaser oscillator 60 is therefore reflected off the dichroic mirror 66toward the mirror 19. On the other hand, the fluorescence produced inthe cancer-related gene products is reflected off the mirror 19, thenpasses through the dichroic mirror 66, and reaches the photodetector 35.The optical part 65 can instead be formed, for example, of a prism or aλ/4 plate.

The imaging unit 10 includes the endoscope 2 and the photodetector 35and applies the laser light L to the interior of the living body (livingcell group) to image the intracellular fluorescence intensity of thefluorescence emitted from the living-body stain and the intracellulardistribution form of the living-body stain, the intracellularfluorescence intensity and the intracellular distribution formreflecting a specific cancer-related gene product expression pattern.The imaging unit 90 includes a focal position control unit and controlsthe focal position control unit to image the cancer-related geneexpression pattern stained with the stain.

The photodetector 35 detects the fluorescence produced when the laserlight L is applied to the living cells and converts the fluorescenceinto an electric signal according to the intensity of the fluorescence.The photodetector 35 can, for example, be a photomultiplier or a CCDsemiconductor image sensor.

The endoscope 2 includes an inner tube 12 and an outer tube 13, whichsurrounds part of the outer surface of the inner tube 12, as shown inFIG. 10 . The inner tube 12 and part of the outer tube 13 are insertedinto the living body. The inner tube 12 has a length, for example, of 50mm and an outer diameter ranging, for example, from 3 to 10 mm. Alinear-motion actuator is attached to the inner tube 12, and the innertube 12 is movable relative to the outer tube 13 in the axial directionX by about 25 mm. An ultrasonic motor is further attached to the innertube 12, and the inner tube 12 is revolvable relative to the outer tube13 over 360°. The action of the inner tube 12 in the axial direction Xor the revolutional direction R is controlled by the control unit 50.

An imaging head 11 is provided at a front-end-side end portion of theinner tube 12 of the endoscope 2. The imaging head 11 is inserted alongwith the inner tube 12 into the living body in such a way that theimaging head 11 and the inner tube 12 pass by the insertion tube 20, asshown in the section (b) of FIG. 9 . The imaging head 11 is socontrolled as to move in the living body based on the actions of theinner tube 12 in the axial direction X and the revolutional direction R.

The imaging head 11 includes the objective lens 16, a focal pointchanger 18, a spacer 17, and the mirror 19.

The mirror 19 is a part that redirects the laser light L outputted fromthe laser oscillator 60 toward the objective lens 16 or redirects thefluorescence emitted from the cancer-related gene products toward thephotodetector 35, as described above.

The objective lens 16 is so provided as to face the inner wall surface113 of the living body. The objective lens 16 to be used can be a lenshaving a diameter ranging from 3 mm to 5 mm, which allows the objectivelens 16 to be readily inserted into the living body.

The focal point changer 18 is, for example, a piezoelectric actuator andmoves the objective lens 16 in the optical axis direction to change theposition of the focal point of the objective lens 16. The focal pointchanger 18 operates under the control of the control unit 50 and canadjust the focal point over a depth range from 0 to 1000 μm below thesurface of the inner wall surface 113.

The spacer 17 has, for example, an annular shape and is provided aroundthe space between the objective lens 16 and the inner wall surface 113.The spacer 17 is a part not only for preventing the objective lens 16from coming into contact with the inner wall of the living body but formaintaining a fixed distance between the objective lens 16 and the innerwall surface 113.

The image processing unit 70 stores the converted electric signal(fluorescence intensity) from the photodetector 35 and the coordinateposition of the imaging unit 10 sent from the control unit 50 with theelectric signal and the coordinate position related to each other andprocesses the data on the electric signal and the coordinate position togenerate a digital image. The generated digital image is, for example,displayed on a monitor, printed out, or recorded on the storage unit 51in the control unit 50. The coordinate position of the imaging unit 10may be expressed, for example, in the form of the distance from areference location on the patient (throat or anus, for example) and theangle of revolution of the imaging head 11.

The control unit 50 is formed, for example, of a CPU, a ROM, and a RAM.The control unit 50 controls the action of the imaging head 11 via theinner tube 12. Specifically, the control unit 50 controls the movementof the imaging head 11 not only in the revolutional direction R alongthe inner circumference of the inner wall of the digestive tract 112 butin the tract longitudinal direction of the digestive tract 112 (alongaxis X of digestive tract). The control unit 50 further changes theposition of the objective lens 16 in the optical axis direction bycontrolling the action of the focal point changer 18 to control theposition where the focus is achieved in the living body. The controlunit 50 can further adjust the laser power by controlling the laseroscillator 60.

The control unit 50 includes the evaluation unit 52, which evaluates thegrade of cancerization, and the storage unit 51, which storesinformation that serves as an evaluation reference when the grade ofcancerization is evaluated, as described above.

The storage unit 51 stores information on the grade of cancerization andinformation on the staining state of the living cell group with the twopieces of information related to each other. The grade of cancerizationis, for example, expressed by the first to fourth stages divided inaccordance with the state of the activity of each of the cancer-relatedgenes, as shown in FIG. 1C. The information on the staining state of theliving cell group contains, for example, the area of or the number ofcells in the stained region (region stained in chromatic color) of theliving cell group in each of the stages described above. The area of orthe number of cells in the stained region varies in accordance with thetype of the stain to be used and therefore needs to be acquired inadvance in accordance with the stain to be used.

The evaluation unit 52 evaluates the grade of cancerization by comparingthe staining state of a captured image with information on the stainingstates stored in the storage unit 51. For example, the evaluation unit52 compares the area of or the number of cells in the stained region inthe captured image with the area of or the number of cells in thestained region in each of the stages that is stored in the storage unit51 to determine the stage to which the staining state of the living cellgroup belongs.

The cancer test device 1 according to the present embodiment includesthe application unit 40, which applies to a living cell group the stain45, which selectively stains a cancer-related gene expression pattern ofthe living cells in a chromatic color, the imaging unit 10, which imagesthe living cell group to which the stain 45 has been applied, and theevaluation unit 52, which evaluates the grade of cancerization of theliving cell group based on the staining state of the living cell groupin the captured image. As image information, in addition to the area ofor the number of cells in the stained region, a difference in theluminance or fluorescence color of the stained region or a difference inthe geometric pattern of the stained region is used. The automaticdiagnosis employs, for example, a method for identifying the expressionlocation based on highly probable luminance and determining the grade ofcancerization based on the state of mixed fluorescence colors. Themethod using the geometric pattern, although the pattern varies on anorgan basis, can be a method for digitizing the density of dark portions(dark hole pattern), a method for digitizing the density ofisland-shaped patterns and recognizing disturbance of the island-shapedpatterns, as described with reference to the section (c) of FIG. 4A andthe section (a) of FIG. 4E, or any other diagnosis method. Further, itis effective to use the interval between the dark portions, thediameters of the dark portions, the interval in the island-shapedpattern, the diameter of the island-shaped pattern, or any othermeasured distance to evaluate and diagnose disturbance of the pattern.

The thus configured cancer test device 1 evaluates the grade ofcancerization based on the staining state of the cancer-related geneexpression patterns of the living cell group, whereby cancerization ofthe living cell group can be grasped in an early stage. Further, sincethe grade of cancerization can be grasped, the prognosis of the cancerpatient can be understood.

Further, the cancer test device 1 can image, out of cancer-related geneexpression patterns stained with the stain 45, a cancer-related geneexpression pattern in a cell population having an average diameter atleast greater than or equal to 0.1 mm but smaller than or equal to 0.4mm. The grade of cancerization of the living cells in the pre-cancerstate can therefore be grasped, whereby the prognosis of the cancerpatient can be understood in the ultra-early stage before the cancercell population 152 clearly manifests. The diameter of a cancer-relatedgene expression pattern is the diameter of a circle having the same areaas the area of the cancer-related gene expression pattern. The averagediameter is calculated by measuring the diameter described above of eachstained cell group present in a fixed area and dividing the diameters bythe number of stained cell groups.

Further, the cancer test device 1 according to the present embodimentcan image a cancer-related gene expression pattern stained with thestain 45 and present over the depth range greater than or equal to 10 μmbut smaller than or equal to 1000 μm below the mucosa surface in aliving body. The grade of cancerization of the interior of the livingbody over the depth range greater than or equal to 10 μm but smallerthan or equal to 1000 below the mucosa surface can therefore be grasped,whereby the cancer cell population 152 can be detected with no missingpart in the ultra-early stage before the cancer cell population appearson the mucosa surface, and the prognosis of the cancer patient can beunderstood (FIG. 19A). For example, cellular morphological fluorescenceimages over a depth range from 10 μm to 50 μm below the mucosa surfaceof a digestive tract can be converted into a full-circumferencepanoramic image around the circumference of the digestive tract, asshown in FIG. 19A. As a result, “cancer detection with no missing part”can be achieved based on the image because the full circumferenceimaging is guaranteed. FIG. 19B shows the endoscope 2 of a cancer testdevice 1A.

The endoscope 2 of the cancer test device 1A includes the inner tube 12and the outer tube 13, which surrounds part of the outer surface of theinner tube 12. A linear-motion actuator is attached to the inner tube12, and the inner tube 12 is movable relative to the outer tube 13 inthe axial direction X. An ultrasonic motor is further attached to theinner tube 12, and the inner tube 12 is revolvable relative to the outertube 13 over 360°. The action of the inner tube 12 in the axialdirection X or the revolutional direction R is controlled by the controlunit 50. The cancer test device 1A allows understanding of the distanceto a lesion in the axial direction X and the angle in the revolutionaldirection R with respect to a predetermined position, for example, theanus in the case of the large intestine and the mouse in the case of thestomach, whereby the position of the lesion can be identified.

[2. Example of Action of Cancer Test Device]

An example of the action of the cancer test device 1 according to thepresent embodiment will next be described. FIG. 12 is a flowchartshowing an example of the action of the cancer test device 1.

A cleaning liquid is first supplied into the closed space S via thesupply port 42 (not shown) before the stain 45 is applied in FIG. 8 .The inner wall surface 113 of the digestive tract 112 is thus cleaned.The cleaning liquid is then sucked and recovered via the recovery port43. A pronase liquid is then supplied via the supply port 42 into theclosed space S. Excess of the mucus having adhered to the inner wallsurface 113 of the digestive tract 112 is thus removed. The pronaseliquid is then sucked and recovered via the recovery port 43.

The application unit 40 then applies the stain 45 containing acurcumin-based compound to a living cell group (S11 a: applicationstep). Specifically, the stain 45 containing a curcumin-based compoundis supplied via the supply port 42 into the closed space S to fill theclosed space S with the stain 45. The stain 45 is then left for 2 to 5minutes, and the space S is then cleaned with the cleaning liquid. TheSTAT3-family cancer-related gene product in the living cell group in thedigestive tract 112 is thus stained with the stain 45 containing acurcumin-based compound.

The application unit 40 containing the stain 45 containing phloxine isthen used to apply the stain 45 containing phloxine to the living cellgroup (S11 b: application step). Specifically, the stain 45 containingphloxine is supplied via the supply port 42 into the closed space S tofill the closed space S with the stain 45. The stain 45 is then left for2 to 5 minutes, and the space S is then cleaned with the cleaningliquid. The ras-family cancer-related gene expression pattern in theliving cell group in the digestive tract 112 is thus stained with thestain 45 containing phloxine. The application of the two types of stain45 causes the living cell group in the inner wall of the digestive tract112 to be double stained.

The imaging unit 10 then images the cancer-related gene expressionpatterns of the living cell group stained with the stains (S12: imagingstep). Specifically, the control unit 50 performs the imaging whilecontrolling the movement of the imaging head 11 not only in therevolutional direction R along the inner wall of the digestive tract 112but in the tract longitudinal direction of the digestive tract 112(along axis X of digestive tract).

The evaluation unit 52 then evaluates the grade of cancerization and theprognosis of the cancer patient based on the staining state in thecaptured image (S13: evaluation step).

Specifically, the area and the number of cells in the stained regionstained with the stains 45 are determined. The area of each of thestained regions is determined by evaluating whether or not the staininghas been performed for each pixel of the captured image based on apredetermined threshold and replacing the number of pixels determined tohave been stained with a corresponding area. The number of cells in eachof the stained regions is determined based on the number of nuclei ofthe cell or the number of compartments separated by the cell membrane inthe stained region. Data on the resultant area or the number of cells isthen compared with data stored in the storage unit 51 and representingthe area or the number of cells to evaluate the grade of cancerizationand the prognosis of the cancer patient.

For example, when the area of the stained region stained with the stain45 containing phloxine is greater than or equal to 0.0075 mm² butsmaller than 3 mm², it may be considered that the expression of theras-family cancer-related gene product has been enhanced, and it maytherefore be determined that the grade of cancerization is the secondstage (see FIG. 1C) or later. For example, when the number of cells inthe stained region stained with the stain 45 containing phloxine isgreater than or equal to 8 but smaller than 512, it may be consideredthat the expression of the ras-family cancer-related gene product hasbeen enhanced, and it may therefore be determined that the grade ofcancerization is the second stage or later. Further, the second stageand the following stages may further be divided into sub-stages for theevaluation of the grade of cancerization and the prognosis of the cancerpatient.

The grade of cancerization may be evaluated based on the state of theexpression of the ras-family cancer-related gene product, as describedabove, but not necessarily. The grade of cancerization may instead beevaluated by using the stain 45 containing a curcumin-based compoundbased on the state of activity of the STAT3-family cancer-related gene.Still instead, a predetermined stain may be used to stain theAPC/β-catenin-family or p53-family cancer-related gene expressionpattern, and whether or not the amplitude of the growth inhibitingsignal has decreased may be examined for evaluation of the grade ofcancerization and the prognosis of the cancer patient.

The multiphoton-laser-microscope-based cancer test device 1 according tothe present embodiment can also be used to remove a cancerous portion(cancer cell population) with the laser light.

For example, in a case where an image produced by the imaging unit 10contains a cancerous portion, the power of the laser light L isincreased as compared with the power in the imaging under themultiphoton laser microscope, and the laser light L having the increasedpower is applied to the cancerous portion to specifically remove(evaporate) only the cancerous portion. The laser power in the removingoperation is 10 to 20 times the power in the imaging operation or rangesfrom 2 to 3 W. The cancerous portion can therefore be reliably removedin an early stage of the cancerous portion.

Second Embodiment

A cancer test device according to a second embodiment is a stationarytest device and used in a case where a patient is externally tested ortissue cells immediately after removed from a patient but within about20 minutes are tested.

A cancer test device 201 according to the present embodiment includes alaser oscillator 213, a beam diameter adjuster 215, a two-dimensionalscanner 217, a dichroic mirror 219, an objective lens 221, a focusedlight depth adjuster 223, a photodetector 225, a fluorescence imagegeneration unit 227, a monitor 229, and a control unit 231, as shown inFIG. 13 .

The laser oscillator 213 to be used is configured to have a pulse widthranging from several tens to several hundreds of femtosecond and a pulserepetition frequency ranging from several tens to several hundreds ofmegahertz and further configured to be capable of adjusting the power ofthe pulse laser light or a CW laser outputting light having a visiblewavelength for a typical confocal laser microscope.

In a case where the pulse laser for the multiphoton laser is used, thebeam diameter adjuster 215 is a beam expander that changes the beamdiameter of the pulse laser light in accordance with a beam diameteradjustment signal from the control unit 231.

The two-dimensional scanner 217 is formed, for example, of twogalvanometric mirrors and changes the position where the pulse laserlight is focused in two axial directions perpendicular to the opticalaxis.

The dichroic mirror 219 separates fluorescence produced in acancer-related gene product in living cells when the living cells areirradiated with the pulse laser light.

The objective lens 221 focuses the pulse laser light emitted from thelaser oscillator 213 in the living cells and focuses the fluorescenceproduced in the cancer-related gene product in the multiphotonabsorption phenomenon. The focused light depth adjuster 223 can move theobjective lens 221 in the optical axis direction based on a controlsignal, so that the focused light position where the laser light isfocused can be adjusted.

The photodetector 225 detects the fluorescence produced in thecancer-related gene product and converts the fluorescence into anelectric signal according to the intensity of the fluorescence.

The scan state of the two-dimensional scanner 217 and the adjustedfocused light position provided by the focused light depth adjuster 223(position in depth direction) are parameters representing thecoordinates of the focused light position, and the fluorescence imagegeneration unit 227 stores the parameters representing the coordinatesand the electric signal (that is, fluorescence intensity) sent from thephotodetector 225 with the parameters and the electric signal related toeach other and processes the data on the parameters and the electricsignal to produce a fluorescence image. The produced fluorescence imageis displayed on the monitor 229.

The control unit 231 includes an action control unit 233, a test pulseintensity setting unit 235, an irradiation range setting unit 239, andan irradiation period setting unit 241. The action control unit 233controls the actions of the laser oscillator 213, the beam diameteradjuster 215, the two-dimensional scanner 217, and the focused lightdepth adjuster 223.

The test pulse intensity setting unit 235 sets pulse laser lightintensity suitable for acquisition of a fluorescence image of acancer-related gene expression pattern to test the living cells.

The irradiation range setting unit 239 sets the range over which theliving cells are irradiated with the pulse laser light. The actioncontrol unit 233 then controls the actions of the two-dimensionalscanner 217 and the focused light depth adjuster 223 to cause the pulselaser light to be radiated and focused at the set depth within the setirradiation range. The irradiation period setting unit 241 sets a periodfor which the living cells are irradiated with the pulse laser light.The action control unit 233 controls the power of the pulse laser lightfrom the laser oscillator 213 to cause the pulse laser light to beradiated only for the set period.

The control unit 231 in the present embodiment includes the same storageunit 51 and evaluation unit 52 as those in the first embodiment. Thatis, the cancer test device 201 evaluates the grade of cancerization andprognosis of the living cell group based on the staining state of theliving cell group in the captured image.

The cancer test device 201, which evaluates the grade of cancerizationbased on the staining state of the cancer-related gene expressionpattern in the living cell group, can grasp the cancerization of theliving cell group in an early stage. Further, the cancer test device201, which allows grasp of the grade of cancerization based on the stateof expression of the cancer-related gene, allows understanding of theprognosis of the cancer patient.

The cancer test device 201 includes a treatment pulse intensity settingunit 237, which can set pulse laser light intensity intense enough todestroy the living cells for treatment thereof. Cancer treatment cantherefore be performed on the found cancer cell population in an earlystage.

In addition to the above, the cancer test device 201 according to thepresent embodiment can be implemented in a variety of other forms.

For example, the beam diameter adjuster 215, the two-dimensional scanner217, an optical system formed of the dichroic mirror 219, the objectivelens 221, and the optical path therebetween, and the focused light depthadjuster 223 may be provided in a laser light radiation head 243, and apatient fixation bench 245, on which a patient lies, and a mover 247 maybe further provided, as shown in FIG. 14 to perform a cancer test.

Further, for example, in a state in which part of a living cell group isscraped off a patient and the scraped living cell group is placed on atray (specimen mount), the cancer test device 201 can be used to imagethe living cell group and evaluate the grade of cancerization. In thiscase, the stain 45 may be applied to the living cell group before theliving cell group is scraped off or after the living cell group isscraped off but before the imaging.

Third Embodiment

[1. Basic Configuration of Cancer Test Device]

The basic configuration of a cancer test device 301 according to a thirdembodiment, which is a case where a CW laser outputting light having avisible wavelength for a typical confocal laser microscope is used, willnext be described with reference to FIGS. 15 to 18 .

FIG. 15 is a schematic view showing a front-end-side end portion of theendoscope 2 of the cancer test device 301 in FIG. 17 . FIG. 16 is aschematic view showing the entire endoscope 2. FIG. 17 is a blockdiagram showing the control configuration of the cancer test device 301.

The cancer test device 301 includes the imaging unit 10, which includesthe endoscope 2, the control unit 50, and the image processing unit 70,as shown in FIG. 17 . The cancer test device 301 further includes alaser oscillator 60C and an optical part 65C. The cancer test device 301still further includes the application unit 40, which supplies a stainto the interior of a living body (see FIG. 8 ).

The laser light L1 emitted from the laser oscillator 60C is reflectedoff a dichroic mirror 66C, which is the optical part 65C, furtherreflected off a mirror 19C in the endoscope 2, and applied to the livingbody. Living cells irradiated with the laser light L1 producefluorescence, and the fluorescence is reflected off the mirror 19C,passes through the dichroic mirror 66C, and is detected with aphotodetector 35C. The light detected with the photodetector 35C isconverted into an electric signal, and the image processing unit 70forms an image according to the electric signal. Since the color of thefluorescence changes in accordance with the stain, the photodetector 35Ccan be formed of a plurality of photodetectors, and a color separationoptical filter can be disposed on the upstream side of the photodetector35C for color separation. The actions, functions, and roles of the partsdescribed above are roughly the same as those in FIG. 11 , but thereference numerals of the parts are suffixed with “C” representing thefirst letter of the CW laser for distinction purposes because a confocallaser microscope differs in principle from a multiphoton lasermicroscope.

The laser oscillator 60C includes a plurality of lasers that allow thewavelength of the laser light L1 to be changed stepwise over awavelength range from 405 to 980 nm, and the wavelength is selected inaccordance with the characteristics of a fluorescence reaction thatoccurs in a measurement target. The lasers may each operate in pulses orcontinuous oscillation. In the case of pulse operation, the operationfrequency is at least several tens of kilohertz, and the duty rangesfrom 5% to 50%. The frequency and duty ranges are so selected inconsideration of the imaging sweep frequency that a sharp image isproduced. The laser light L1 in the present embodiment is confocal laserlight, and the laser oscillator 60C uses, for example, a laser capableof emitting light having an intensity peak wavelength of 488 nm, 594 nm,or 647 nm and a power of 30 mW at the maximum. The power of the laserlight emitted from the laser in the imaging operation ranges from 5 to10 mW, but not necessarily. The laser oscillator 60C can adjust theintensity of the laser light L1 in accordance with the degree ofstaining and the degree of the intensity of fluorescence.

The dichroic mirror 66C, which is the optical part 65C, reflects lighthaving the same wavelength as that of the laser light L1 and transmitslight having the other wavelengths. The laser light L1 emitted from thelaser oscillator 60C is therefore reflected off the dichroic mirror 66Ctoward the mirror 19C. On the other hand, the fluorescence produced inthe living cells is reflected off the mirror 19C, then passes throughthe dichroic mirror 66C, and reaches the photodetector 35C. The opticalpart 65C can instead be formed, for example, of a prism or a λ/4 plate.

The imaging unit 10 includes the endoscope 2 and the photodetector 35Cand applies the laser light L1 to the interior of the living body toimage the cell form in the living body.

The photodetector 35C detects the fluorescence produced when the laserlight L1 is applied to the living cells and converts the fluorescenceinto an electric signal according to the intensity of the fluorescence.The photodetector 35C can, for example, be a photomultiplier or a CCDsemiconductor image sensor. A pinhole or any other component is providedas a part that provides the confocal laser function.

The endoscope 2 includes the inner tube 12 and the outer tube 13, whichsurrounds part of the outer surface of the inner tube 12, as shown inFIG. 16 . The inner tube 12 and part of the outer tube 13 are insertedinto the living body. The inner tube 12 has a length, for example, of 50mm and an outer diameter ranging, for example, from 3 to 10 mm. Alinear-motion actuator is attached to the inner tube 12, and the innertube 12 is movable relative to the outer tube 13 in the axial directionX by about 25 mm. An ultrasonic motor is further attached to the innertube 12, and the inner tube 12 is revolvable relative to the outer tube13 over 360°. The action of the inner tube 12 in the axial direction Xor the revolutional direction R is controlled by the control unit 50.

An imaging head 11C is provided at a front-end-side end portion of theinner tube 12 of the endoscope 2. The imaging head 11C is inserted alongwith the inner tube 12 into the living body in such a way that theimaging head 11C and the inner tube 12 pass by the insertion tube 20, asshown in FIG. 15 . The imaging head 11C is so controlled as to move inthe living body based on the actions of the inner tube 12 in the axialdirection X and the revolutional direction R.

The imaging head 11C includes an objective lens 16C, the focal pointchanger 18, the spacer 17, and the mirror 19C.

The mirror 19C is a part that redirects the laser light L1 outputtedfrom the laser oscillator 60C toward the objective lens 16C or redirectsthe fluorescence emitted from the living cells toward the photodetector35C, as described above.

The objective lens 16C is so provided as to face the inner wall surface113 of the living body. The objective lens 16C has, for example, adiameter of 10 mm, a magnification of 10 times, a resolution of 5 μm,and an imaging field of view of 3 mm×3 mm. The objective lens 16Cinstead has a diameter of 12 mm, a magnification of 40 times, aresolution of 10 μm, and a field of view of 7.5 mm×7.5 mm. The wider theimaging field of view, the better. The objective lens 16C may stillinstead be so configured that part of a lens having either of thediameters described above is cut or the diameter of the objective lens16C is reduced to a value ranging from 3 mm to 5 mm so that theobjective lens is readily inserted into the living body with the sameresolution maintained. The objective lens 16C may be so disposed as toincline with respect to the inner wall surface 113. Performing theimaging with the objective lens 16C inclining allows the cell forms ofthe epithelium 120 and the gland 130 to be simultaneously observed.

The focal point changer 18 is, for example, a piezoelectric actuator oran electromagnetic actuator and moves the objective lens 16C in theoptical axis direction to change the position of the focal point of theobjective lens 16C. The focal point changer 18 operates under thecontrol of the control unit 50 and can adjust the focal point over adepth range from 0 to 75 μm below the inner wall surface (mucosasurface) 113. Changing the position of the focal point allows imaging ofthe state of the living body at a predetermined depth below the innerwall surface 113 of the digestive tract 112.

The spacer 17 has, for example, an annular shape and is provided aroundthe space between the objective lens 16C and the inner wall surface 113.The spacer 17 is a part not only for preventing the objective lens 16Cfrom coming into contact with the inner wall of the living body but formaintaining a fixed distance between the objective lens 16C and theinner wall surface 113. The distance between the objective lens 16C andthe inner wall surface (mucosa surface) 113 is set at an appropriatevalue, for example, a value greater than or equal to 1 mm but smallerthan or equal to 10 mm by exchanging the spacer 17 to another before theimaging starts or adding a distance changeable mechanism using anactuator or any other device. The control unit 50 controls the movementof the imaging head 11C (inner tube 12) with the spacer 17 being incontact with the inner wall surface 113 and maintains the fixed distancefrom the objective lens 16C to the inner wall surface 113.

The control unit 50 is formed, for example, of a CPU, a ROM, and a RAM.The control unit 50 controls the action of the imaging head 11 via theinner tube 12. Specifically, the control unit 50 controls the movementof the imaging head 11C not only in the circumferential direction alongthe inner circumference of the inner wall of the digestive tract 112 butin the tract longitudinal direction of the digestive tract 112 (alongaxis of digestive tract). The control unit 50 further changes theposition of the objective lens 16C in the optical axis direction bycontrolling the action of the focal point changer 18 to control theposition where the focus is achieved in the living body. The controlunit 50 can further adjust the laser power by controlling the laseroscillator 60C.

The image processing unit 70 stores the converted electric signal(fluorescence intensity) from the photodetector 35C and the coordinateposition of the imaging unit 10 sent from the control unit 50 with theelectric signal and the coordinate position related to each other andprocesses the data on the electric signal and the coordinate position togenerate a digital image. The generated digital image is, for example,displayed on a monitor, printed out, or recorded on a storage device.The coordinate position of the imaging unit 10 may be expressed, forexample, in the form of the distance from a reference location on thepatient (throat or anus, for example) and the angle of revolution of theimaging head 11C.

The confocal-laser-endoscope-based cancer test device 301 according tothe present embodiment includes the imaging unit 10, which includes theimaging head 11C, which is inserted into a living body, and images theliving body by applying the laser light to the living body via theimaging head 11C, and the control unit 50, which controls the operationof the imaging head 11C. The imaging head 11C includes the objectivelens 16C and the focal point changer 18, which is capable of changingthe position of the focal point of the objective lens 16C in the depthdirection of the living body, and the control unit 50 causes the focalpoint changer 18 to operate in such a way that the position of the focalpoint has a predetermined depth deeper than or equal to 10 μm butshallower than or equal to 100 μm (desirably deeper than or equal to 10μm but shallower than or equal to 70 μm) below the mucosa surface in theliving body. The imaging unit 10 applies the laser light to a cell grouppresent in the living body and stained with the stain 45, whichselectively stains the cell group in a chromatic color, and images thestained cell group at the predetermined depth.

A method for controlling the focal point with a fixed positionalrelationship between the objective lens 16C and the mucosa surfacemaintained will be described. Reference character 171 in FIG. 17 denotesa second laser oscillator, which emits continuous parallelized light asreference light L2, for example, having a wavelength of 680 nm and apower of about 5 mW. A beam splitter, a half-silvered mirror, or anyother component causes the light from the second laser oscillator 171 totravel along the optical path of the light from the laser oscillator60C. In FIG. 17 , the optical path of the reference light L2 is drawnwith a broken line slightly shifted from the optical path of the lightfrom the laser oscillator 60C for ease of understanding. The referencelight L2 described above travels along roughly the same path as that ofthe cancer-test laser light L1 but travels along a different opticalpath beyond a beam splitter 173 and enters a focal point control opticalunit 174. In a case where the position of the focal point of theobjective lens 16C changes, a cylindrical lens, a beam splitter, andother components form an optical part configuration capable of detectingthe amount of change in the position of the focal point. Referencecharacter 175 denotes a photodetector that is typically divided into 2or 4 blocks. The light detected with the thus configured photodetectoris converted, for example, with a differential amplifier into anelectric signal proportional to a change in the positional relationshipbetween the objective lens 16C and the mucosa surface. The control ofthe position of the objective lens described above is used, for example,in an optical disk device and is fully applicable to an endoscopedevice. To apply the position control described above to a cancer testdevice, a point to be aware of is that the laser light L1 for imagingand the reference light L2 preferably have different wavelengths so thatthe two light beams are readily separated from each other. Separatingthe wavelengths of the two light beams from each other by at least 100nm achieves optical characteristics of the imaging system and the focalpoint control system that allow the two light beams to be satisfactorilyseparate from each other. In the case where the focal point controlsystem described above is provided, applying bias voltage to the controlsystem allows fine adjustment of the position of the focal point.Changing the bias voltage stepwise allows automatic control of theposition where the laser light L1 is focused in the depth direction.

The transmittance or reflectance of the optical parts 11C, 35C, 65C,66C, 172, 173, and 174 greatly depends on the wavelength of the laserlight beams L1 and L2. Modularizing the optical parts in accordance withthe wavelengths of the laser light beams and preparing a plurality ofmodules can therefore readily handle a situation in which thewavelengths of the laser light beams are changed in accordance with thestain to be used or a body site to be tested.

As described above, even the cancer test device 301 including theconfocal laser endoscope can acquire images at the depths deeper than orequal to 10 μm but shallower than or equal to 70 μm below the inner wallsurface (mucosa surface) 113 of the living body. As a result, a lesioncan be readily found, and selecting the wavelengths and the intensitiesof the laser light beams allows acquisition of images with no load onthe patient, such as optical damage of cells irradiated with the laserlight.

[2. Action of Cancer Test Device]

The action of the cancer test device 301 according to the presentembodiment will next be described. FIG. 18 is a flowchart showing anexample of the action of the cancer test device 301. In the cancer testdevice 301 according to the present embodiment, the two different stains45 are applied to a living cell group to stain two cancer-related geneexpression patterns in colors different from each other, and the stainedcancer-related gene expression patterns are imaged.

A cleaning liquid is first supplied into the closed space S via thesupply port 42 (not shown) before the stains 45 are applied. The innerwall surface 113 of the digestive tract 112 is thus cleaned. Thecleaning liquid is then sucked and recovered via the recovery port 43. Apronase liquid is then supplied via the supply port 42 into the closedspace S. Excess of the mucus having adhered to the inner wall surface113 of the digestive tract 112 is thus removed. The pronase liquid isthen sucked and recovered via the recovery port 43.

The application unit 40 then applies the stain 45 containing acurcumin-based compound to the living cell group (S11 a: applicationstep). Specifically, the stain 45 containing a curcumin-based compoundis supplied via the supply port 42 into the closed space S to fill theclosed space S with the stain 45. The stain 45 is then left for 2 to 5minutes, and the space S is then cleaned with the cleaning liquid. TheSTAT3-family cancer-related gene product in the living cell group in thedigestive tract 112 is thus stained with the stain 45 containing acurcumin-based compound.

The application unit 40 containing the stain 45 containing phloxine isthen used to apply the stain 45 containing phloxine to the living cellgroup (S11 b: application step). Specifically, the stain 45 containingphloxine is supplied via the supply port 42 into the closed space S tofill the closed space S with the stain 45. The stain 45 is then left for2 to 5 minutes, and the space S is then cleaned with the cleaningliquid. The ras-family cancer-related gene expression pattern in theliving cell group in the digestive tract 112 is thus stained with thestain 45 containing phloxine.

The imaging unit 10 then images the cancer-related gene expressionpatterns of the living cell group stained with the two stains 45described above (S12: imaging step). Specifically, the cancer-relatedgene expression patterns stained in the two different colors areirradiated with excitation light beams having two different wavelengthsto allow the imaging unit 10 to image the plurality of cancer-relatedgene expression patterns. In the present embodiment, the laser light L1having a wavelength of 488 nm is radiated as the excitation light beamsfor causing the cancer-related gene expression pattern stained with thestain 45 containing a curcumin-based compound to emit fluorescence, andthe laser light L1 having a wavelength of 594 nm is radiated as theexcitation light beams for causing the cancer-related gene expressionpattern stained with the stain 45 containing phloxine to emitfluorescence. The cancer-related gene expression patterns stained in thetwo colors are then sequentially irradiated with the two types of laserlight L1, and fluorescence produced by the irradiation is detected withthe photodetector 35C.

The evaluation unit 52 then evaluates the grade of cancerization and theprognosis of the cancer patient based on the staining states in thecaptured image (S13: evaluation step).

As described above, the cancer-related gene expression patterns stainedwith the two stains 45 are each irradiated with excitation light beamsaccording to the corresponding stain 45 for detection of the state ofthe activity of the plurality of cancer-related genes and evaluation ofthe grade of cancerization. Further, irradiating each of thecancer-related gene expression patterns with the laser light L1 having asingle wavelength as described above allows stable detection of thefluorescence emitted from the cancer-related gene expression pattern.

The above description has been made with reference to the case where thetwo stains 45 and excitation light beams corresponding thereto are usedto detect two cancer-related gene expression patterns, but notnecessarily. Three or more stains 45 and excitation light beamscorresponding thereto may be used to detect three or more cancer-relatedgene expression patterns. The stain 45 is not limited to acurcumin-based compound or phloxine and may instead be High Red V80,sulfuretin, erythrosine, epigallocatechin-gallate, indocyanine green,malvidin, β-carotene, High Red BL, 6-gingerol, myricetin, tricenidine,or petunidine.

For example, use of the stain 45 that stains the cancer-related geneexpression pattern of the APC/β-catenin family or the p53 family, whichis a brake-system gene, allows examination of whether or not theamplitude of the cell growth inhibiting signal has decreased. Therefore,a brake-system gene can be detected, and the prognosis of the cancerpatient can be evaluated.

As described above, staining cancer-related gene expression patternswith many stains 45 and irradiating each of the stained cancer-relatedgene expression patterns with excitation light beams according to thecorresponding stain 45 allows detection of the many cancer-relatedgenes. The grade of cancerization can therefore be analyzed from diverseviewpoints, whereby the probability of evaluation of the prognosis canbe increased.

Determination of a cancerous site and a healthy site based on thepermeability difference of the edible pigments between these two siteswill next be described.

A case where a cell form in a living body is imaged under themultiphoton laser microscope (FV1000MPE manufactured by OlympusCorporation) while changing the depth of the focal point and a pluralityof captured images are cut in a predetermined position to create across-sectional image (tomographic image) will be described withreference to FIGS. 20A, 20B, and 20C. A laboratory mouse was used as theliving body.

FIGS. 20A to 20C show images illustrating the cell form over apredetermined depth range below the inner wall surface (mucosa surface),specifically, three-dimensional data images obtained by performingimaging at 2-μm intervals from the mucosa surface (depth 0) to a depthof 150 μm and layering the captured 75 images in total on one another.In each of FIGS. 20A to 20C, the section (a) shows an image of the cellgroup in a plan view viewed in the direction perpendicular to the innerwall surface 113, the section (b) shows a cross-sectional image of thesection (a) taken along the line b-b, and the section (c) shows across-sectional image of the section (a) taken along the line c-c.

As the stain for staining the cell group, both the stain containingcurcumin and the stain containing Acid Red (red #106) were used. Thestaining period was set at 5 minutes. The staining period is the periodfor which the stain is caused to be in contact with the cell group andthe dye of the stain is allowed to penetrate into the cells themselvesor the gaps between the cells.

FIGS. 20A to 20C show images obtained by imaging the same cell group atthe same time and filtering the images to extract different colors(wavelengths). FIG. 20A shows images representing the extracted colorregion stained both with the curcumin dye and the Acid Red dye. FIG. 20Bshows images representing the extracted color region stained with thecurcumin dye. FIG. 20C shows images representing the extracted colorregion stained with the Acid Red dye. FIGS. 20A to 20C showmonochromatic images that are inherently color images, and thedifference in the tendency of the staining performed by the stainscauses the region stained with the curcumin dye to be displayed in agreen fluorescent color and the region stained with the Acid Red dye tobe displayed in a pale red or a near orange fluorescent color, wherebythe difference in color is more distinctively expressed.

FIGS. 20A to 20C show cancer tissue and healthy mucosa tissue andindicate that the dyes differ from each other in terms of permeability.The curcumin dye shows higher permeability in the cancer tissue than inthe healthy mucosa tissue, as shown in FIG. 20B. Specifically, in thecase of the curcumin dye, the depth to which the tissue is stained isabout 40 μm in the cancer tissue, whereas the depth is about 20 μm inthe healthy mucosa tissue. The Acid Red dye shows lower permeability inthe cancer tissue than in the healthy mucosa tissue, as shown in FIG.20C. Specifically, in the case of the Acid Red dye, the depth to whichthe tissue is stained is about 40 μm in the cancer tissue, whereas thedepth is about 70 μm in the healthy mucosa tissue.

As described above, the permeability of a dye varies depending onwhether the cell form is cancer tissue or healthy mucosa tissue. It isbelieved based on the characteristic described above that measurement ofthe depth to which a cell group displayed in a cross-sectional image isstained allows identification of the cell group, a healthy cell group ora cancer cell group. The cancer test device performs depth directioncontrol below the cell surface at the time of imaging to evaluatewhether the cell group displayed in the cross-sectional image issuspicious of a lesion based on a depth to which the cell group has beenstained. For example, when the depth to which the cell group has beenstained with the curcumin dye is (at least 1.5 times, for example)greater than the depth to which the healthy mucosa tissue has beenstained with the curcumin dye, the control unit 50 determines thatcancer cells have developed, whereas when the two depths are similar toeach other (difference is smaller than 1.5 times, for example), thecontrol unit 50 determines that no cancer cells have developed. Further,when the depth to which the cell group has been stained with the AcidRed dye is (at least 0.6 times, for example) smaller than the depth towhich the healthy mucosa tissue has been stained with the Acid Red dye,the control unit 50 determines that cancer cells have developed, whereaswhen the two depths are similar to each other (difference is at least0.6 times, for example), the control unit 50 determines that no cancercells have developed. It is noted that after whether or not cancer cellsare present is evaluated based, for example, on single or doublestaining, the evaluation described above based on a cross-sectionalimage increases the reliability of the evaluation. In the presentexample, curcumin, which stains the STAT3-family cancer-related geneproduct with a high degree of penetration, and Acid Red, whichpenetrates into a healthy cell by a high degree, are used. Staining asample with phloxine, erythrosine, or any other stain that penetratesinto a ras-family cancer gene product with a high degree of penetrationallows comparison between the degrees of progress of the STAT3 familyand the ras family in terms of the degree of stain penetration.

Other Examples

The cancer test devices 1, 201, and 301 according to the embodiments ofthe present invention have been described, but the present invention isnot limited to the embodiments described above and variations thereof.For example, aspects in which the embodiments described above andvariations thereof are changed as follows may also fall within the scopeof the present invention.

For example, in the first embodiment, to stain a cancer-related geneexpression pattern, stains are used one by one for sequentiallystaining, but not necessarily. A plurality of dyes may be mixed with oneanother in advance to produce a mixed stain containing the plurality ofdyes, and the mixed stain may be used to perform simultaneous staining.Further, an oral cleaning liquid containing a mucosa cleaning agent, astain, and other substances may be used to stain a digestive tract. Inthe above description, the staining has been described in two ways: Acancer-related gene expression pattern is stained; and a cancer-relatedgene product is stained. The cancer-related gene expression pattern isnot only a result of the operation of staining a cancer-related geneproduct but a state in which the cancer-related gene product havinggrown to some extent is the observation target.

In the first embodiment, a living body is stained with two differentcolor stains before the imaging, but not necessarily. A living body maybe stained with three or more different color stains before the imaging.

In the first and second embodiments, multiphoton laser light is used aslaser light for the cancer test device 1, but not necessarily, andconfocal laser light can instead be used. Still instead, a typical CWlaser microscope or a single-color fluorescence microscope may also beused as long as the wavelength to be used is appropriately selected.From the viewpoint of depth-direction imaging and resolution, it isdesirable to use multiphoton laser light having a wavelength rangingfrom 600 nm to 1600 nm. However, achieving the analysis described aboveto some extent under a confocal laser microscope using a wavelengthranging from 400 to 700 nm or a fluorescence microscope using a typicalCW laser that emits light having a wavelength ranging from 400 to 700 nmor single-color light having a wavelength ranging from 400 to 700 nm byskillfully selecting a lens or a wavelength to achieve magnification orresolution that allows the nucleus of a stained cell to be viewed fallswithin the scope of the present invention. Further, changing thewavelength of the laser light with which a sample is irradiated andchanging a filter for image formation in accordance with the wavelengthof the fluorescence emitted from a stain have been described above.

The cancer test devices 1, 201, and 301 according to the first andsecond embodiments are also applicable to luminal organs (such asbronchus, urinary bladder, and urinary duct) other than digestivetracts, and can further visualize the kidney, the liver, the brain, theretina, and other cell structures although there is a restriction on thevisualization range of 1 mm or smaller in depth below the surface of thesample.

Further, the images described above are not limited to still images andmay instead be motion images or a combination of still images and motionimages. For example, motion images can be captured in preparatorydiagnosis, a postoperative regular test, and other occasions, whereasstills image can be used in precise diagnosis. The enlargementmagnification scale at the time of imaging is also not limited to fallwithin the ranges described above.

Cell tissue to be stained in living body staining may be an in-vivo celltissue or fresh ex-vivo cell tissue within 20 minutes immediately afterthe cell tissue is surgically excised or otherwise separated out of theliving body.

Further, the cancer test devices described above each have beendescribed on the assumption that the cancer test device includes thestaining unit, the imaging unit including an endoscope, the storageunit, and the evaluation unit, but the cancer test device does notnecessarily include both the staining unit and the evaluation unit. Aconfiguration in which another device performs the staining and aconfiguration in which the content in the storage unit is shared withanother device or a computer and the other device or the computerperforms the analysis and evaluation fall within the scope of thepresent invention. Further, the imaging unit does not necessarilyinclude an endoscope depending on a site to be analyzed and may insteadinclude a microscope having a fixed objective lens, as the specificationdescribes both the cases described above. The latter case therefore, ofcourse, falls within the scope of the present invention.

INDUSTRIAL APPLICABILITY

The cancer test devices according to the present invention are used tofind cancer in an early stage in the digestive tract, the respiratorysystem, the renal/urinary system, the utero-ovarian reproductive system,the cerebrospinal nervous system, and other body sites.

EXPLANATION OF SIGNS

-   1, 1A, 201, 301 Cancer test device-   2 Endoscope-   10 Imaging unit-   11, 11C Imaging head-   12 Inner tube-   13 Outer tube-   16, 16C Objective lens-   17 Spacer-   18 Focal point changer-   19, 19C Mirror-   20 Insertion tube-   21 First balloon-   22 Second balloon-   35, 35C Photodetector-   40 Application unit-   42 Supply port-   43 Recovery port-   45 Stain-   50, 231 Control unit-   51 Storage unit-   52 Evaluation unit-   60, 60C Laser oscillator-   65, 65C Optical part-   66, 66C Dichroic mirror-   67 Two-dimensional scanner-   70 Image processing unit-   112 Digestive tract-   113 Inner wall surface of digestive tract (mucosa surface)-   120 Epithelium-   121 Epithelial cell-   125 Nucleus of epithelial cell-   126 Cytoplasm of epithelial cell-   130 Gland-   131 Glandular cell-   132 Capillary-   133 Connective tissue-   135 Nucleus of glandular cell-   136 Cytoplasm of glandular cell-   137 Basement membrane-   138 Crypt-   152 Cancer cell population-   160 Muscular layer of mucosa-   L, L1 Laser-   L2 Reference light-   S Closed space

The invention claimed is:
 1. A cancer test device comprising: anapplication unit that applies a stain that selectively stains acancer-related gene product of living cells in a chromatic color to aliving cell group, wherein, the cancer-related gene product isSTAT3-family cancer-related gene product that transmits a signal thatpromotes growth of the living cells and the stain is a curcumin-basedcompound; an imaging unit that images the living cell group to which thestain has been applied; and an evaluation unit that evaluates a grade ofcancerization of the living cell group and diversity of cancer-relatedgene expression based on a staining state of the living cell group in animage produced by the imaging.
 2. The cancer test device according toclaim 1, wherein the evaluation unit performs the evaluation based on anarea of a stained region of the living cell group.
 3. The cancer testdevice according to claim 1, wherein the evaluation unit performs theevaluation based on number of stained cells in a stained region of theliving cell group.
 4. The cancer test device according to claim 1,wherein the evaluation unit performs the evaluation based on number andaverage diameter of stained cell groups in a fixed area containing astained region of the living cell group.
 5. The cancer test deviceaccording to claim 1, wherein the imaging unit images the living cellgroup to which the stain has been applied by irradiating it withmultiphoton or confocal laser light.
 6. The cancer test device accordingto claim 1, wherein the imaging unit images the cancer-related geneexpression pattern stained with the stain and having a diameter largerthan or equal to 0.1 mm but smaller than or equal to 0.4 mm.
 7. Thecancer test device according to claim 1, wherein the application unitstains a plurality of the cancer-related gene expression patterns incolors different from one another by applying a plurality of thedifferent stains to the living cell group, and the imaging unit images aplurality of the cancer-related gene expression patterns by irradiatingthe plurality of cancer-related gene expression patterns stained in thedifferent colors with a plurality of excitation light beams according tothe stains.
 8. The cancer test device according to claim 7, wherein thestains are formed of at least two types of stain, and the excitationlight beams with which the plurality of cancer-related gene expressionpatterns are irradiated are selected in correspondence with the types ofthe stain.
 9. The cancer test device according to claim 1, wherein theimaging unit includes a focal point position control unit and controlsthe focal point position control unit to image the cancer-related geneexpression pattern presenting at a depth deeper than or equal to 10 μmbut shallower than or equal to 1000 μm below a surface in the livingbody stained with the stain.
 10. The cancer test device according toclaim 9, wherein the focal point position control unit is controlled tochange a focal point at fixed intervals from the surface in the sameimaging position in the living body stained with the stain to performimaging at different-depth focal point positions, a plurality ofcaptured images is superimposed on one another in a focal point positioninformation order into a stereoscopic image, and the evaluation isperformed based on a degree of penetration of the stain in thestereoscopic image.
 11. The cancer test device according to claim 1,wherein the application unit applies the stain containing acurcumin-based compound to the living cell group and then applies astain that stains a ras-family cancer-related gene product thattransmits a signal that promotes growth of the living cells, wherein thestain that stains a ras-family cancer-related gene product is phloxine,erythrosine, merbromin, fast green FCF, or meclocycline sulfosalicylateto the living cell group.